Methods to express recombinant proteins from lentiviral vectors

ABSTRACT

Lentivector constructs for expression of recombinant proteins, polypeptides or fragments thereof and methods of making the same are described. The lentivectors typically have a self-processing cleavage sequence between a first and second protein or polypeptide coding sequence allowing for expression of a functional protein or polypeptide under operative control of a single promoter and may further include an additional proteolytic cleavage sequence which provides a means to remove the self-processing cleavage sequence from the expressed protein or polypeptide. The vector constructs find utility in methods relating to enhanced production of biologically active proteins, such as immunoglobulins or fragments thereof in vitro and in vivo.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of co-pending U.S.application Ser. No. 11/488,568, filed Jul. 18, 2006, which claims thepriority benefit of U.S. Provisional Patent Application No. 60/700,298,filed Jul. 19, 2005. The priority application is expressly incorporatedby reference herein in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to novel lenti vector constructs designed toexpress recombinant full-length proteins or fragments thereof. The lenticonstructs may be used for ex vivo or in vivo expression of aheterologous protein coding sequence by a cell or organ, or in vitro forthe production of recombinant proteins by lenti vector-transduced cells.

2. Background of the Technology

Recombinant proteins as therapeutic modalities have found increasing usein recent years. Numerous recombinant protein-based therapies are invarious stages of clinical development. One limitation to widespreadclinical application of recombinant protein technology is the difficultyin production of proteins that include two or more coding sequences ordomains such that the domains are expressed in the proper ratio withappropriate post-translational processing resulting in production of afunctional heterodimeric molecule. A further limitation is the high costassociated with the ability to produced adequate levels of protein forclinical applications.

Monoclonal antibodies have been proven as effective therapeutics forcancer and other diseases. Current antibody therapy often involvesrepeat administration and long term treatment regimens, which areassociated with a number of disadvantages, such as inconsistent serumlevels, limited duration of efficacy per administration such thatfrequent readministration is required and high cost. The use ofantibodies as diagnostic tools and therapeutic modalities has also foundincreasing use in recent years. One limitation to the widespreadclinical application of antibody technology is that typically largeamounts of antibody are required for therapeutic efficacy and the costsassociated with production are significant. Chinese Hamster Ovarian(CHO) cells, SP20 and NSO2 myeloma cells are the most commonly usedmammalian cell lines for commercial scale production of glycosylatedhuman proteins such as antibodies. The yields obtained from mammaliancell line production typically range from 50-250 mg/L for 5-7 dayculture in a batch fermentor or 300-1000 mg/L for 7-12 day cultures infed batch fermentors. High-level production often relies upon geneamplification and selection of best performing clones that is timeconsuming and further increases the cost of development and production.In addition, stability issues with respect to antibody-producing celllines are often evident following multiple passages.

Previous attempts to express full length recombinant proteins with twoor more domains or chains (and thus two or more coding sequences or openreading frames (ORFs)) via recombinant DNA technology have met withlimited success, typically resulting in unequal levels of expression ofthe two or more domains or chains of the protein or polypeptide and moreimportantly, a lower level of expression for the second coding sequence.In order to obtain optimal expression of a fully functional andbiologically active protein or polypeptide that has two or more domains,substantially equimolar expression of the two or more domains isrequired. Conventional vectors that rely on dual promoter regulation ofgene expression are invariably affected by promoter interaction (i.e.,promoter interference) that may compromise equimolar or substantiallyequimolar expression of the genes.

Lentiviral vectors are a type of retroviral vector that can infect bothdividing and non-dividing cells. They can be used to express proteinfrom non-dividing or terminally differentiated cells such as neurons,macrophages, hematopoietic stem cells, retinal photoreceptors, muscleand liver cells, cell types for which other vector systems cannot beused effectively.

There remains a need for improved gene expression systems for productionof recombinant proteins and polypeptides, in particular proteins andpolypeptides that have two or more domains or chains, such thatsufficient expression of a biologically active recombinant protein orpolypeptide is achieved at commercially reasonable cost.

The present invention addresses this need by demonstrating thefeasibility and use of lentivector constructs for the expression offunctional recombinant proteins and polypeptides which have two or moredomains.

SUMMARY OF THE INVENTION

The present invention provides lentivector constructs for expression ofprotein or polypeptide open reading frames from a single cell andmethods of using the same.

In one preferred approach, the vectors have a self-processing cleavagesequence between the protein or polypeptide coding sequences allowingfor expression of more than one functional protein or polypeptide usinga single promoter. The invention finds utility in production of two ormore proteins or polypeptides or a protein or polypeptide having two ormore domains (or chains) using a lentiviral vector where sustainedexpression occurs in a single cell. Exemplary lentivector constructscomprise a self-processing cleavage sequence and may further comprise anadditional proteolytic cleavage site for removal of the self-processingcleavage sequence from the expressed protein or polypeptide. The vectorconstructs find utility in methods relating to enhanced production ofbiologically active proteins, polypeptides or fragments thereof, invitro and in vivo.

The invention relates to engineered lentiviral vectors that encode twoor more domains or chains of a multimeric protein. In one aspect themultimeric protein is an immunoglobulin (i.e., an antibody) andfull-length antibody heavy and light chain coding sequences areexpressed using a lentivector comprising a single open reading framedriven by a single promoter wherein the vector comprises a selfprocessing cleavage site or sequence between the heavy and light chaincoding sequences. In another aspect the protein is a multimeric proteinand the full-length coding sequences are expressed using a lentivectorcomprising a single open reading frame driven by a single promoterwherein the vector comprises a self-processing cleavage site orsequence.

In yet another aspect, the invention relates to a method for high levelexpression of recombinant protein using more than one engineeredlentiviral vector wherein each lentivector encodes a single open readingframe of a multimeric protein driven by a single promoter. For example,for expression of a full-length antibody, individual lentivectors thatencode the full-length antibody heavy and light chain, respectively, areused to infect the same cell such that high level expression of abiologically active antibody results.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic depiction of the process for expression of afull-length immunoglobulin (antibody) using constructs that include aself-processing cleavage site, such as 2A, and a furin cleavage site.

FIG. 2 is a schematic depiction of plasmid, lentivirus and AAVexpression cassettes comprising a self-processing cleavage site (2A) forexpression of immunoglobulin heavy (H) and light (L) chains operativelylinked to a CAG promoter (CAG), wherein the vector may further includean additional proteolytic cleavage site (Furin; “F”).

FIG. 3 is a schematic depiction of the time line associated fordevelopment of antibody-expressing clones with an illustration ofapproximate clone development timelines, indicating the advantage oflentivectors over the use of plasmids (transfection and selection) interms of time and antibody expression level.

FIG. 4 illustrates the expression of a full length rat anti-VEGFR2monoclonal antibody (CAG-DC101 IgG1) in different cell lines (CHOD-,HuH7 and PerC6) following transduction with a lentivector, wherein 2Arefers to expression of DC101 via a single vector including a selfprocessing sequence.

FIG. 5 shows the results of a Southern blot analysis to determine thenumber of integrated genomic copies of the lentiviral vector for two 5×transfected clones expressing approximately 20-40 pg/cell/day of DC101antibody. Samples containing known genomic quantities of the lentiviralvector were used as a standard for determining the number of integratedgenomic copies.

FIG. 6 illustrates the antibody expression levels for 60 individualclones isolated from cells that had been transfected 4×, 7× or 9× withthe lentiviral 2A DC101-encoding vector. The amount of antibody produced(in pg/cell/day) is plotted against the number of clones that expressthat amount of recombinant DC101 antibody from each population.

FIG. 7 illustrates the expression of a full length human CMV anti-KDRIgG1 monoclonal antibody in different cell lines (CHOD-, HuH7 and PerC6)following transduction with a lentivector, wherein 2A refers toexpression of the heavy and light chain of KDR via a single vectorincluding a self processing sequence.

FIG. 8 illustrates the results of mass spectral analysis of purifies IgGprotein produced following transfection of an HF2AL plasmid encodinginto CHO cells, followed by furin cleavage and treatment withcarboxypeptidase.

FIG. 9 demonstrates the results of a stability study of thirteenpancreatic clones transduced with a lentiviral vector encoding humanGM-CSF. The GM-CSF expression level of clones expressing 500-2500 ng/10⁶cells/24 hr GM-CSF was maintained in continuous culture for 12 weekswith GM-CSF expression levels tested at 3-week intervals.

FIG. 10 demonstrates the results of a stability study of CT26 clonestransduced with a lentiviral vector encoding murine GM-CSF, where theGM-CSF expression level of the CT26 rodent cell lines was shown to bestable for at least 9 weeks of continuous culture.

DETAILED DESCRIPTION OF THE INVENTION

The various compositions and methods of the invention are describedbelow. Although particular compositions and methods are exemplifiedherein, it is understood that any of a number of alternativecompositions and methods are applicable and suitable for use inpracticing the invention. It will also be understood that an evaluationof the protein or polypeptide expression constructs (vectors) andmethods of the invention may be carried out using procedures standard inthe art.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, molecular biology(including recombinant techniques), microbiology, biochemistry andimmunology, which are known to those of skill in the art. Suchtechniques are explained fully in the literature, such as, MolecularCloning: A Laboratory Manual, second edition (Sambrook et al., 1989);Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture(R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press,Inc.); Handbook of Experimental Immunology (D. M. Weir & C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller & M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis et al., eds., 1994); and Current Protocols inImmunology (J. E. Coligan et al., eds., 1991).

DEFINITIONS

Unless otherwise indicated, all terms used herein have the same meaningas they would to one skilled in the art and the practice of the presentinvention will employ, conventional techniques of microbiology andrecombinant DNA technology, which are within the knowledge of those ofskill of the art.

The term “vector”, as used herein, refers to a DNA or RNA molecule suchas a plasmid, virus or other vehicle, which contains one or moreheterologous or recombinant DNA sequences and is designed for transferbetween different host cells. The terms “expression vector” and “genetherapy vector” refer to any vector that is effective to incorporate andexpress heterologous DNA fragments in a cell. A cloning or expressionvector may comprise additional elements, for example, the expressionvector may have two replication systems, thus allowing it to bemaintained in two organisms, for example in human cells for expressionand in a prokaryotic host for cloning and amplification. Any suitablevector can be employed that is effective for introduction of nucleicacids into cells such that protein or polypeptide expression results,e.g. a viral vector or non-viral plasmid vector. Any cells effective forexpression, e.g., insect cells and eukaryotic cells such as yeast ormammalian cells are useful in practicing the invention.

The terms “heterologous DNA” and “heterologous RNA” refer to nucleotidesthat are not endogenous (native) to the cell or part of the genome inwhich they are present. Generally heterologous DNA or RNA is added to acell by transduction, infection, transfection, transformation or thelike, as further described below. Such nucleotides generally include atleast one coding sequence, but the coding sequence need not beexpressed. The term “heterologous DNA” may refer to a “heterologouscoding sequence” or a “transgene”.

As used herein, the terms “protein” and “polypeptide” may be usedinterchangeably and typically refer to “proteins” and “polypeptides” ofinterest that are expresses using the self processing cleavagesite-containing vectors of the present invention. Such “proteins” and“polypeptides” may be any protein or polypeptide useful for research,diagnostic or therapeutic purposes, as further described below.

The term “replication defective” as used herein relative to a viral genetherapy vector of the invention means the viral vector cannotindependently further replicate and package its genome. For example,when a cell of a subject is infected with rAAV virions, the heterologousgene is expressed in the infected cells, however, due to the fact thatthe infected cells lack AAV rep and cap genes and accessory functiongenes, the rAAV is not able to replicate.

As used herein, a “retroviral transfer vector” refers to an expressionvector that comprises a nucleotide sequence that encodes a transgene andfurther comprises nucleotide sequences necessary for packaging of thevector. Preferably, the retroviral transfer vector also comprises thenecessary sequences for expressing the transgene in cells.

As used herein, “packaging system” refers to a set of viral constructscomprising genes that encode viral proteins involved in packaging arecombinant virus. Typically, the constructs of the packaging systemwill ultimately be incorporated into a packaging cell.

As used herein, a “second generation” lentiviral vector system refers toa lentiviral packaging system that lacks functional accessory genes,such as one from which the accessory genes, vif, vpr, vpu and nef, havebeen deleted or inactivated. See, e.g., Zufferey et al., 1997, Nat.Biotechnol. 15:871-875.

As used herein, a “third generation” lentiviral vector system refers toa lentiviral packaging system that has the characteristics of a secondgeneration vector system, and further lacks a functional tat gene, suchas one from which the tat gene has been deleted or inactivated.Typically, the gene encoding rev is provided on a separate expressionconstruct. See, e.g., Dull et al., 1998, J. Virol. 72(11):8463-8471.

As used herein, “pseudotyped” refers to the replacement of a nativeenvelope protein with a heterologous or functionally modified envelopeprotein.

The term “operably linked” as used herein relative to a recombinant DNAconstruct or vector means nucleotide components of the recombinant DNAconstruct or vector are functionally related to one another foroperative control of a selected coding sequence. Generally, “operablylinked” DNA sequences are contiguous, and, in the case of a secretoryleader, contiguous and in reading frame. However, enhancers do not haveto be contiguous.

As used herein, the term “gene” or “coding sequence” means the nucleicacid sequence which is transcribed (DNA) and translated (mRNA) into apolypeptide in vitro or in vivo when operably linked to appropriateregulatory sequences. The gene may or may not include regions precedingand following the coding region, e.g. 5′ untranslated (5′ UTR) or“leader” sequences and 3′ UTR or “trailer” sequences, as well asintervening sequences (introns) between individual coding segments(exons).

A “promoter” is a DNA sequence that directs the binding of RNApolymerase and thereby promotes RNA synthesis, i.e., a minimal sequencesufficient to direct transcription. Promoters and corresponding proteinor polypeptide expression may be cell-type specific, tissue-specific, orspecies specific. Also included in the nucleic acid constructs orvectors of the invention are enhancer sequences that may or may not becontiguous with the promoter sequence. Enhancer sequences influencepromoter-dependent gene expression and may be located in the 5′ or 3′regions of the native gene.

“Enhancers” are cis-acting elements that stimulate or inhibittranscription of adjacent genes. An enhancer that inhibits transcriptionalso is termed a “silencer”. Enhancers can function (i.e., can beassociated with a coding sequence) in either orientation, over distancesof up to several kilobase pairs (kb) from the coding sequence and from aposition downstream of a transcribed region.

A “regulatable promoter” is any promoter whose activity is affected by acis or trans acting factor (e.g., an inducible promoter, such as anexternal signal or agent).

A “constitutive promoter” is any promoter that directs RNA production inmany or all tissue/cell types at most times, e.g., the human CMVimmediate early enhancer/promoter region which promotes constitutiveexpression of cloned DNA inserts in mammalian cells.

The terms “transcriptional regulatory protein”, “transcriptionalregulatory factor” and “transcription factor” are used interchangeablyherein, and refer to a nuclear protein that binds a DNA response elementand thereby transcriptionally regulates the expression of an associatedgene or genes. Transcriptional regulatory proteins generally binddirectly to a DNA response element, however in some cases binding to DNAmay be indirect by way of binding to another protein that in turn bindsto, or is bound to a DNA response element.

As used herein, the term “sequence identity” means nucleic acid or aminoacid sequence identity between two or more aligned sequences, whenaligned using a sequence alignment program. The terms “% homology” and“% identity” are used interchangeably herein and refer to the level ofnucleic acid or amino acid sequence identity between two or more alignedsequences, when aligned using a sequence alignment program. For example,80% homology means the same thing as 80% sequence identity determined bya defined algorithm under defined conditions.

The terms “identical” or percent “identity” in the context of two ormore nucleic acid or protein sequences, refer to two or more sequencesor subsequences that are the same or have a specified percentage ofamino acid residues or nucleotides that are the same, when compared andaligned for maximum correspondence, as measured using one of thesequence comparison algorithms described herein, e.g. the Smith-Watermanalgorithm, or by visual inspection.

As used herein, an “internal ribosome entry site” or “IRES” refers to anelement that promotes direct internal ribosome entry to the initiationcodon, such as ATG, of a cistron (a protein encoding region), therebyleading to the cap-independent translation of the gene. See, e.g.,Jackson R J, Howell M T, Kaminski A (1990) Trends Biochem Sci15(12):477-83) and Jackson R J and Kaminski, A. (1995) RNA1(10):985-1000. The examples described herein are relevant to the use ofany IRES element, which is able to promote direct internal ribosomeentry to the initiation codon of a cistron. “Under translational controlof an IRES” as used herein means that translation is associated with theIRES and proceeds in a cap-independent manner.

A “self-processing cleavage site” or “self-processing cleavage sequence”is defined herein as a post-translational or co-translational processingcleavage site or sequence. Such a “self-processing cleavage” site orsequence refers to a DNA or amino acid sequence, exemplified herein by a2A site, sequence or domain or a 2A-like site, sequence or domain. Asused herein, a “self-processing peptide” is defined herein as thepeptide expression product of the DNA sequence that encodes aself-processing cleavage site, sequence or domain, which upontranslation mediates rapid intramolecular (cis) cleavage of a protein orpolypeptide comprising the self-processing cleavage site to yielddiscrete mature protein or polypeptide products.

As used herein, the term “additional proteolytic cleavage site”, refersto a sequence which is incorporated into an expression construct of theinvention adjacent a self-processing cleavage site, such as a 2A or 2Alike sequence, and provides a means to remove additional amino acidsthat remain following cleavage by the self processing cleavage sequence.Exemplary “additional proteolytic cleavage sites” are described hereinand include, but are not limited to, furin cleavage sites with theconsensus sequence RXK(R)R (SEQ ID NO: 10). Such furin cleavage sitescan be cleaved by endogenous subtilisin-like proteases, such as furinand other serine proteases within the protein secretion pathway.

As used herein, the terms “immunoglobulin” and “antibody” may be usedinterchangeably and refer to intact immunoglobulin or antibody moleculesas well as fragments thereof, such as Fa, F (ab′)2, and Fv, which arecapable of binding an antigenic determinant. Such an “immunoglobulin”and “antibody” is composed of two identical light polypeptide chains ofmolecular weight approximately 23,000 daltons, and two identical heavychains of molecular weight 53,000-70,000. The four chains are joined bydisulfide bonds in a “Y” configuration. Heavy chains are classified asgamma (IgG), mu (IgM), alpha (IgA), delta (IgD) or epsilon (IgE) and arethe basis for the class designations of immunoglobulins, whichdetermines the effector function of a given antibody. Light chains areclassified as either kappa or lambda. When reference is made herein toan “immunoglobulin or fragment thereof”, it will be understood that sucha “fragment thereof” is an immunologically functional immunoglobulinfragment.

The term “humanized antibody” refers to an antibody molecule in whichone or more amino acids of the antigen binding regions of a non-humanantibody have been replaced in order to more closely resemble a humanantibody, while retaining the binding activity of the original non-humanantibody. See, e.g., U.S. Pat. No. 6,602,503.

The term “antigenic determinant”, as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. Numerous regions of a protein or fragment of aprotein may induce the production of antibodies that binds specificallyto a given region of the three-dimensional structure of the protein.These regions or structures are referred to as antigenic determinants.An antigenic determinant may compete with the intact antigen (i.e., theimmunogen used to elicit the immune response) for binding to anantibody.

The term “fragment”, when referring to a recombinant protein orpolypeptide of the invention means a polypeptide which has an amino acidsequence which is the same as part of but not all of the amino acidsequence of the corresponding full length protein or polypeptide, andwhich retains at least one of the functions or activities of thecorresponding full length protein or polypeptide. The fragmentpreferably includes at least 20-100 contiguous amino acid residues ofthe full-length protein or polypeptide.

The terms “administering” or “introducing”, as used herein refer todelivery of a vector for recombinant protein expression to a cell or tocells and/or organs of a subject. Such administering or introducing maytake place in vivo, in vitro or ex vivo. A vector for recombinantprotein or polypeptide expression may be introduced into a cell bytransfection, which typically means insertion of heterologous DNA into acell by physical means (e.g., calcium phosphate transfection,electroporation, microinjection or lipofection); infection, whichtypically refers to introduction by way of an infectious agent, i.e. avirus; or transduction, which typically means stable infection of a cellwith a virus or the transfer of genetic material from one microorganismto another by way of a viral agent (e.g., a bacteriophage).

“Transformation” is typically used to refer to bacteria comprisingheterologous DNA or cells that express an oncogene and have thereforebeen converted into a continuous growth mode such as tumor cells. Avector used to “transform” a cell may be a plasmid, virus or othervehicle.

Typically, a cell is referred to as “transduced”, “infected”,“transfected” or “transformed” dependent on the means used foradministration, introduction or insertion of heterologous DNA (i.e., thevector) into the cell. The terms “transduced”, “transfected” and“transformed” may be used interchangeably herein regardless of themethod of introduction of heterologous DNA. A cell may be “transduced”by infection with a viral vector.

As used herein, the terms “stably transformed”, “stably transfected” and“transgenic” refer to cells that have a non-native (heterologous)nucleic acid sequence integrated into the genome. Stable transfection isdemonstrated by the establishment of cell lines or clones comprised of apopulation of daughter cells containing the transfected DNA stablyintegrated into their genomes. In some cases, “transfection” is notstable, i.e., it is transient. In the case of transient transfection,the exogenous or heterologous DNA is expressed, however, the introducedsequence is not integrated into the genome and is considered to beepisomal.

As used herein, “ex vivo administration” refers to a process whereprimary cells are taken from a subject, a vector is administered to thecells to produce transduced, infected or transfected recombinant cellsand the recombinant cells are readministered to the same or a differentsubject.

A “multicistronic transcript” refers to an mRNA molecule that containsmore than one protein coding region, or cistron. An mRNA comprising twocoding regions is denoted a “bicistronic transcript”. The “5′-proximal”coding region or cistron is the coding region whose translationinitiation codon (usually AUG) is closest to the 5′-end of amulticistronic mRNA molecule. A “5′-distal” coding region or cistron isone whose translation initiation codon (usually AUG) is not the closestinitiation codon to the 5′ end of the mRNA. The terms “5′-distal” and“downstream” are used synonymously to refer to coding regions that arenot adjacent to the 5′ end of an mRNA molecule.

As used herein, “co-transcribed” means that two (or more) coding regionsor polynucleotides are under transcriptional control of a singletranscriptional control or regulatory element.

The term “host cell”, as used herein refers to a cell that has beentransduced, infected, transfected or transformed with a vector. Thevector may be a plasmid, a viral particle, a phage, etc. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothose skilled in the art. It will be appreciated that the term “hostcell” refers to the original transduced, infected, transfected ortransformed cell and progeny thereof.

As used herein, the terms “biological activity” and “biologicallyactive”, refer to the activity attributed to a particular protein in acell line in culture or in a cell-free system, such as a ligand-receptorassay in ELISA plates. The “biological activity” of an “immunoglobulin”,“antibody” or fragment thereof refers to the ability to bind anantigenic determinant and thereby facilitate immunological function.

As used herein, the terms “tumor” and “cancer” refer to a cell thatexhibits a loss of growth control and forms unusually large clones ofcells. Tumor or cancer cells generally have lost contact inhibition andmay be invasive and/or have the ability to metastasize.

Internal Ribosome Entry Site (IRES)

IRES elements were first discovered in picornavirus mRNAs (Jackson R J,Howell M T, Kaminski A (1990) Trends Biochem Sci 15(12):477-83) andJackson R J and Kaminski, A. (1995) RNA 1(10):985-1000). Examples ofIRES generally employed by those of skill in the art include thosedescribed in U.S. Pat. No. 6,692,736. Examples of “IRES” known in theart include, but are not limited to IRES obtainable from picornavirus(Jackson et al., 1990) and IRES obtainable from viral or cellular mRNAsources, such as for example, immunoglobulin heavy-chain binding protein(BiP), the vascular endothelial growth factor (VEGF) (Huez et al. (1998)Mol. Cell. Biol. 18(11):6178-6190), the fibroblast growth factor 2(FGF-2), and insulin-like growth factor (IGFII), the translationalinitiation factor eIF4G and yeast transcription factors TFIID and HAP4,the encephelomycarditis virus (EMCV) which is commercially availablefrom Novagen (Duke et al. (1992) J. Virol 66(3):1602-9) and the VEGFIRES (Huez et al. (1998) Mol Cell Biol 18(11):6178-90). IRES have alsobeen reported in different viruses such as cardiovirus, rhinovirus,aphthovirus, HCV, Friend murine leukemia virus (FrMLV) and Moloneymurine leukemia virus (MoMLV). As used herein, “IRES” encompassesfunctional variations of IRES sequences as long as the variation is ableto promote direct internal ribosome entry to the initiation codon of acistron. An IRES may be mammalian, viral or protozoan.

The IRES promotes direct internal ribosome entry to the initiation codonof a downstream cistron, leading to cap-independent translation. Thus,the product of a downstream cistron can be expressed from a bicistronic(or multicistronic) mRNA, without requiring either cleavage of apolyprotein or generation of a monocistronic mRNA. Internal ribosomeentry sites are approximately 450 nucleotides in length and arecharacterized by moderate conservation of primary sequence and strongconservation of secondary structure. The most significant primarysequence feature of the IRES is a pyrimidine-rich site whose start islocated approximately 25 nucleotides upstream of the 3′ end of the IRES.See Jackson et al.(1990).

In eukaryotic cells, translation is normally initiated by the ribosomescanning from the capped mRNA 5′ end, under the control of initiationfactors. However, several cellular mRNAs have been found to have IRESstructure to mediate the cap-independent translation (van der Velde, etal. (1999) Int J Biochem Cell Biol. 31:87-106. An IRES sequence may betested and compared to a 2A sequence as shown in Example 1. In oneexemplary protocol a test vector or plasmid is generated with onetransgene, such as PF-4 or VEGF-TRAP, placed under translational controlof an IRES, 2A or 2A-like sequence to be tested. A cell is transfectedwith the vector or plasmid containing the IRES- or 2A-reporter genesequences and an assay is performed to detect the presence of thetransgene. In one illustrative example, the test plasmid comprisesco-transcribed PF-4 and VEGF-TRAP coding sequences transcriptionallydriven by a CMV promoter wherein the PF-4 or VEGF-TRAP coding sequenceis translationally driven by the IRES, 2A or 2A-like sequence to betested. Host cells are transiently transfected with the test vector orplasmid by means known to those of skill in the art and assayed for theexpression of the transgene.

For some time, in order to express two or more proteins from a singleviral or non-viral vector, an internal ribosome entry site (IRES)sequence has been commonly used to drive expression of the second,third, fourth gene, etc. Although the use of IRES is considered to bethe state of the art by many, when two genes are linked via IRES, theexpression level of the second gene is often significantly reduced(Furler et al., Gene Therapy 8:864-873 (2001)). In fact, the use of anIRES to control transcription of two or more genes operably linked tothe same promoter can result in lower level expression of the second,third, etc. gene relative to the gene adjacent the promoter. Inaddition, an IRES sequence may be sufficiently long to present issueswith the packaging limit of the vector, e.g., the eCMV IRES has a lengthof 507 base pairs.

The present invention provides advantages over the use of an IRES inthat a vector for recombinant protein or polypeptide expressioncomprising a self-processing peptide (exemplified herein by 2A peptides)facilitates expression of two or more protein or polypeptide codingsequences using a single promoter, wherein the two or more proteins orpolypeptides are expressed in a substantially equimolar ratio.

Self-Processing Cleavage Sites or Sequences

The linking of proteins in the form of polyproteins in a single openreading frame is a strategy adopted in the replication of many virusesincluding picornaviridae. Upon translation, virus-encoded proteinasesmediate rapid intramolecular (cis) cleavage of a polyprotein to yielddiscrete mature protein products. Foot and Mouth Disease viruses (FMDV)are a group within the picomaviridae that express a single, long openreading frame encoding a polyprotein of approximately 225 kD. The fulllength translation product undergoes rapid intramolecular (cis) cleavageat the C-terminus of a self-processing cleavage site, for example, a 2Asite or region, located between the capsid protein precursor (P1-2A) andreplicative domains of the polyprotein 2BC and P3, with the cleavagemediated by proteinase-like activity of the 2A region itself (Ryan etal., J. Gen. Virol. 72:2727-2732, 1991); Vakharia et al., J. Virol.61:3199-3207, 1987). Similar domains have also been characterized fromaphthoviridea and cardioviridae of the picornavirus family (Donnelly etal., J. Gen. Virol. 78:13-21, 1997).

A “self-processing cleavage site” or “self-processing cleavage sequence”as defined above refers to a DNA or amino acid sequence, wherein upontranslation, rapid intramolecular (cis) cleavage of a polypeptidecomprising the self-processing cleavage site occurs to result inexpression of discrete mature protein or polypeptide products. Such a“self-processing cleavage site”, may also be referred to as apost-translational or co-translational processing cleavage site,exemplified herein by a 2A site, sequence or domain. It has beenreported that a 2A site, sequence or domain demonstrates a translationaleffect by modifying the activity of the ribosome to promote hydrolysisof an ester linkage, thereby releasing the polypeptide from thetranslational complex in a manner that allows the synthesis of adiscrete downstream translation product to proceed (Donnelly, 2001).Alternatively, a 2A site, sequence or domain demonstrates“auto-proteolysis” or “cleavage” by cleaving its own C-terminus in cisto produce primary cleavage products (Furler; Palmenberg, Ann Rev.Microbiol. 44:603-623 (1990)).

Although the mechanism is not part of the invention, the activity of a2A-like sequence may involve ribosomal skipping between codons whichprevents formation of peptide bonds (de Felipe et al., Human GeneTherapy 11:1921-1931 (2000); Donnelly et al., J. Gen. Virol.82:1013-1025 (2001)), although it has been considered that the domainacts more like an autolytic enzyme (Ryan et al., Virol. 173:35-45(1989). Studies in which the Foot and Mouth Disease Virus (FMDV) 2Acoding region was cloned into expression vectors and transfected intotarget cells showed FMDV 2A cleavage of artificial reporter polyproteinsin wheat-germ lysate and transgenic tobacco plants (Halpin et al., U.S.Ser. No. 5,846,767; 1998 and Halpin et al., The Plant Journal17:453-459, 1999); Hs 683 human glioma cell line (de Felipe et al., GeneTherapy 6:198-208, 1999); hereinafter referred to as “de Felipe II”);rabbit reticulocyte lysate and human HTK-143 cells (Ryan et al., EMBO J.13:928-933 (1994)); and insect cells (Roosien et al., J. Gen. Virol.71:1703-1711, 1990). The FMDV 2A-mediated cleavage of a heterologouspolyprotein has been shown for IL-12 (p40/p35 heterodimer; Chaplin etal., J. Interferon Cytokine Res. 19:235-241, 1999). The referencedemonstrates that in transfected COS-7 cells, FMDV 2A mediated thecleavage of a p40-2A-p35 polyprotein into biologically functionalsubunits p40 and p35 having activities associated with IL-12.

The FMDV 2A sequence has been incorporated into retroviral vectors,alone or combined with different IRES sequences to constructbicistronic, tricistronic and tetracistronic vectors. The efficiency of2A-mediated gene expression in animals was demonstrated by Furler (2001)using recombinant adeno-associated viral (AAV) vectors encodingα-synuclein and EGFP or Cu/Zn superoxide dismutase (SOD-1) and EGFPlinked via the FMDV 2A sequence. EGFP and α-synuclein were expressed atsubstantially higher levels from vectors which included a 2A sequencerelative to corresponding IRES-based vectors, while SOD-1 was expressedat comparable or slightly higher levels. Furler also demonstrated thatthe 2A sequence results in bicistronic gene expression in vivo afterinjection of 2A-containing AAV vectors into rat substantia nigra.

For the present invention, the DNA sequence encoding a self-processingcleavage site is exemplified by viral sequences derived from apicornavirus, including but not limited to an entero-, rhino-, cardio-,aphtho- or Foot-and-Mouth Disease Virus (FMDV). In one preferredembodiment, the self-processing cleavage site coding sequence is derivedfrom a FMDV. Self-processing cleavage sites include but are not limitedto 2A and 2A-like sites, sequences or domains (Donnelly et al., J. Gen.Virol. 82:1027-1041 (2001). Positional subcloning of a 2A sequencebetween two or more heterologous DNA sequences in a vector constructallows the delivery and expression of two or more open reading frames byoperable linkage to a single promoter. Preferably, self-processingcleavage sites such as FMDV 2A sequences provide a unique means toexpress and deliver from a single viral vector, two or more proteins,polypeptides or peptides which can be individual parts of for example,an immunoglobulin Factor VIII, a cytokine, or another heterodimericprotein, an antibody, or a heterodimeric receptor.

FMDV 2A is a polyprotein region that functions in the FMDV genome todirect a single cleavage at its own C-terminus, thus functioning in cis.The FMDV 2A domain is typically reported to be about nineteen aminoacids in length ((LLNFDLLKLAGDVESNPGP (SEQ ID NO: 1);TLNFDLLKLAGDVESNPGP (SEQ ID NO: 2); Ryan et al., J. Gen. Virol.72:2727-2732 (1991)), however oligopeptides of as few as fourteen aminoacid residues ((LLKLAGDVESNPGP (SEQ ID NO: 3)) have also been shown tomediate cleavage at the 2A C-terminus in a fashion similar to its rolein the native FMDV polyprotein processing.

Variations of the 2A sequence have been studied for their ability tomediate efficient processing of polyproteins (Donnelly M L L et al.2001). Homologues and variant 2A sequences are included within the scopeof the invention and include but are not limited to the sequencespresented in Table 1, below:

TABLE 1 Table of Exemplary 2A Sequences (SEQ ID) SEQUENCE NO: 1LLNFDLLKLAGDVESNPGP NO: 2 TLNFDLLKLAGDVESNPGP NO: 3 LLKLAGDVESNPGP NO: 4NFDLLKLAGDVESNPGP NO: 5 QLLNFDLLKLAGDVESNPGP NO: 6APVKQTLNFDLLKLAGDVESNPGP NO: 7 VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP NO: 8 LLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP NO: 9EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP

Distinct advantages of self-processing cleavage sequences, such as a 2Asequence or a variant thereof are their use in generating vectorsexpressing self-processing polyproteins. This invention includeslentivectors which comprise the coding sequence for two or more proteinsor polypeptides linked via self-processing cleavage sites such that theindividual proteins or polypeptides are expressed in equimolar or closeto equimolar amounts following the cleavage of the polyprotein due tothe presence of the self-processing cleavage site. These proteins may beheterologous to the vector itself, to each other or to theself-processing cleavage site, e.g., FMDV. Thus the self-processingcleavage sites for use in practicing the invention do not discriminatebetween heterologous proteins or polypeptides and coding sequencesderived from the same source as the self-processing cleavage site, inthe ability to function or mediate cleavage.

The expression levels of individual proteins, polypeptides or peptidesfrom a promoter driving a single open reading frame comprising more thantwo coding sequences are closer to equimolar as compared to expressionlevels achievable using IRES sequences or dual promoters. Elimination ofdual promoters reduces promoter interference that may result in reducedand/or impaired levels of expression for each coding sequence.

In one preferred embodiment, the FMDV 2A sequence included in alentivector according to the invention encodes amino acid residuescomprising LLNFDLLKLAGDVESNPGP (SEQ ID NO: 1). Alternatively, alentivector according to the invention may encode amino acid residuesfor other 2A-like regions as discussed in Donnelly et al., J. Gen.Virol. 82:1027-1041 (2001) and including but not limited to a 2A-likedomain from picornavirus, insect virus, Type C rotavirus, trypanosomerepeated sequences or the bacterium, Thermatoga maritima.

The invention contemplates the use of nucleic acid sequence variantsthat encode a self-processing cleavage site, such as a 2A or 2A-likepolypeptide, and nucleic acid coding sequences that have a differentcodon for one or more of the amino acids relative to that of the parent(native) nucleotide. Such variants are specifically contemplated andencompassed by the present invention. Sequence variants ofself-processing cleavage peptides and polypeptides are included withinthe scope of the invention as well.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, JMol. Biol. 48: 443 (1970), by the search for similarity method ofPearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), by the BLAST algorithm, Altschulet al., J Mol. Biol. 215: 403-410 (1990), with software that is publiclyavailable through the National Center for Biotechnology Information(www.ncbi.nlm.nih.gov/), or by visual inspection (see generally, Ausubelet al., infra) For purposes of the present invention, optimal alignmentof sequences for comparison is most preferably conducted by the localhomology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981).See, also, Altschul, S. F. et al., 1990 and Altschul, S. F. et al.,1997.

In accordance with the present invention, also encompassed are sequencevariants which encode self-processing cleavage polypeptides andpolypeptides themselves that have 80, 85, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99% or more sequence identity to the native sequence.

A nucleic acid sequence is considered to be “selectively hybridizable”to a reference nucleic acid sequence if the two sequences specificallyhybridize to one another under moderate to high stringency hybridizationand wash conditions. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe. Forexample, “maximum stringency” typically occurs at about Tm-5° C. (5°below the Tm of the probe); “high stringency” at about 5-10° below theTm; “intermediate stringency” at about 10-20° below the Tm of the probe;and “low stringency” at about 20-25° below the Tm. Functionally, maximumstringency conditions may be used to identify sequences having strictidentity or near-strict identity with the hybridization probe; whilehigh stringency conditions are used to identify sequences having about80% or more sequence identity with the probe.

Moderate and high stringency hybridization conditions are well known inthe art (see, Sambrook, et al., 1989, Chapters 9 and 11, and in Ausubel,F. M., et al., 1993. An example of high stringency conditions includeshybridization at about 42° C. in 50% formamide, 5×SSC, 5× Denhardt'ssolution, 0.5% SDS and 100 μg/ml denatured carrier DNA followed bywashing 2× in 2×SSC and 0.5% SDS at room temperature and two additionaltimes in 0.1×SSC and 0.5% SDS at 42° C. 2A sequence variants that encodea polypeptide with the same biological activity as the 2A polypeptidesdescribed herein and hybridize under moderate to high stringencyhybridization conditions are considered to be within the scope of thepresent invention.

As a result of the degeneracy of the genetic code, a number of codingsequences can be provided which encode the same protein, polypeptide orpeptide, such as 2A or a 2A-like peptide. For example, the triplet CGTencodes the amino acid arginine Arginine is alternatively encoded byCGA, CGC, CGG, AGA, and AGG. Therefore it is appreciated that suchsubstitutions in the coding region fall within the sequence variantsthat are covered by the present invention.

Removal of Self-Processing Peptide Sequences

One concern associated with the use of self-processing peptides, such asa 2A or 2A-like sequence is that the C terminus of the expressedpolypeptide contains amino acids derived from the self-processingpeptide, i.e. 2A-derived amino acid residues. These amino acid residuesare “foreign” to the host and may elicit an immune response when therecombinant protein is expressed in vivo (i.e., expressed from a viralor non-viral vector in the context of gene therapy or administered as anin vitro-produced recombinant protein or polypeptide) or delivered invivo following in vitro or ex vivo expression. In addition, if notremoved, self-processing peptide-derived amino acid residues mayinterfere with protein secretion in producer cells and/or alter proteinconformation, resulting in a less than optimal expression level and/orreduced biological activity of the recombinant protein.

The invention includes lenti-based expression constructs, engineeredsuch that an additional proteolytic cleavage site is provided between afirst protein or polypeptide coding sequence (the first or 5′ ORF) andthe self processing cleavage site as a means for removal of selfprocessing cleavage site derived amino acid residues that are present inthe expressed protein product.

Examples of additional proteolytic cleavage sites are furin cleavagesites with the consensus sequence RXK(R)R (SEQ ID NO: 10), which can becleaved by endogenous subtilisin-like proteases, such as furin and otherserine proteases. The inventors have demonstrated that self-processing2A amino acid residues at the C terminus of a first expressed proteincan be efficiently removed by introducing a furin cleavage site RAKR(SEQ ID NO: 18) between the first polypeptide and a self-processing 2Asequence. In addition, use of a plasmid containing a 2A sequence and afurin cleavage site adjacent to the 2A sequence was shown to result in ahigher level of protein expression than a plasmid containing the 2Asequence alone. This improvement provides a further advantage in thatwhen 2A amino acid residues are removed from the C-terminus of theprotein, longer 2A- or 2A like sequences or other self-processingsequences can be used. See, e.g., U.S. Patent Publication Nos.20040265955 and 20050003482, expressly incorporated by reference herein.

It is often advantageous to produce therapeutic proteins, polypeptides,fragments or analogues thereof with fully human characteristics. Thesereagents avoid the undesired immune responses induced by proteins,polypeptides, fragments or analogues thereof originating from differentspecies. To address possible host immune responses to amino acidresidues derived from self-processing peptides, the coding sequence fora proteolytic cleavage site may be inserted (using standard methodologyknown in the art) between the coding sequence for a first protein andthe coding sequence for a self-processing peptide so as to remove theself-processing peptide sequence from the expressed protein orpolypeptide. This finds particular utility in therapeutic and diagnosticproteins and polypeptides for use in vivo.

Any additional proteolytic cleavage site known in the art that can beexpressed using recombinant DNA technology may be employed in practicingthe invention. Exemplary additional proteolytic cleavage sites which canbe inserted between a polypeptide or protein coding sequence and a selfprocessing cleavage sequence include, but are not limited to:

a). Furin consensus sequence or site: RXK(R)R (SEQ ID. NO:10);

b). Factor Xa cleavage sequence or site: IE(D)GR (SEQ ID. NO:11);

c). Signal peptidase I cleavage sequence or site: e.g., LAGFATVAQA (SEQID. NO: 12); and

d). Thrombin cleavage sequence or site: LVPRGS (SEQ ID. NO: 13).

As detailed herein, the 2A peptide sequence provides a “cleavage” sitethat facilitates the generation of both chains of an immunoglobulin orother protein during the translation process. In one exemplaryembodiment, the C-terminus of the first protein, for example theimmunoglobulin heavy chain, contains approximately 13 amino acidresidues that are derived from the 2A sequence itself. The number ofresidual amino acids is dependent upon the 2A sequence used. As setforth above, when a furin cleavage site sequence, e.g., RAKR (SEQ ID NO:18), is inserted between the first protein and the 2A sequence, the 2Aresidues are removed from the C-terminus of the first protein. However,mass spectrum data indicates that the C-terminus of the first proteinexpressed from the RAKR-2A construct contains two additional amino acidresidues, RA, derived from the furin cleavage site RAKR (SEQ ID NO: 18).

In one embodiment, the invention provides a method for removal of theseresidual amino acids and a composition for expression of the same. Anumber of novel constructs have been designed that provide for removalof these additional amino acids from the C-terminus of the protein.Furin cleavage occurs at the C-terminus of the cleavage site, which hasthe consensus sequence RXR(K)R (SEQ ID NO: 19), where X is any aminoacid. In one aspect, the invention provides a means for removal of thenewly exposed basic amino acid residues R or K from the C-terminus ofthe protein by use of an enzyme selected from a group of enzymes calledcarboxypeptidases (CPs), which include, but not limited to,carboxypeptidase D, E and H (CPD, CPE, CPH). Since CPs are able toremove basic amino acid residues at the C-terminus of a protein, allamino acid resides derived from a furin cleavage site which containexclusively basic amino acids R or K, such as RKKR (SEQ ID NO: 14), RKRR(SEQ ID NO: 15), RRRR (SEQ ID NO: 17), etc., can be removed by a CP. Aseries of immunoglobulin expression constructs that contain a 2Asequence and a furin cleavage site and which have basic amino acidresidues at the C terminus have been constructed to evaluate efficiencyof cleavage and residue removal. An exemplary construct design is thefollowing: H chain—furin (e.g, RKKR (SEQ ID NO: 14), RKRR (SEQ ID NO:15), RRKR (SEQ ID NO: 16) or RRRR (SEQ ID NO: 17))-2A-L chain or Lchain—furin (e.g, RKKR (SEQ ID NO: 14), RKRR (SEQ ID NO: 15), RRKR (SEQID NO: 16) or RRRR (SEQ ID NO: 17))-2A-H chain A schematic depiction ofexemplary constructs is provided in FIGS. 14 and 15, respectively ofU.S. Ser. No. 60/659,871, expressly incorporated by reference herein.

As will be apparent to those of skill in the art, there is a basic aminoacid residue (K) at the C terminus of the immunoglobulin heavy (H) chain(rendering it subject to cleavage with carboxypeptidase), while theimmunoglobulin light (L) chain, terminates with a non-basic amino acidC. In one preferred embodiment of the invention, an antibody expressionconstruct comprising a furin site and a 2A sequence is provided whereinthe immunoglobulin L chain is 5′ to the immunoglobulin H chain such thatfollowing translation, the additional furin amino acid residues arecleaved with carboxypeptidase.

Immunoglobulins and Fragments Thereof

Antibodies are immunoglobulin proteins that are heterodimers of a heavyand light chain and have proven difficult to express in a full-lengthform from a single vector in mammalian culture expression systems. Threemethods are currently used for production of vertebrate antibodies, invivo immunization of animals to produce “polyclonal” antibodies, invitro cell culture of B-cell hybridomas to produce monoclonal antibodies(Kohler, et al., Eur. J. Immunol., 6: 511, 1976; Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, 1988) and recombinantDNA technology (described for example in Cabilly et al., U.S. Pat. No.6,331,415).

The basic molecular structure of immunoglobulin polypeptides is known toinclude two identical light chains with a molecular weight ofapproximately 23,000 daltons, and two identical heavy chains with amolecular weight 53,000-70,000, where the four chains are joined bydisulfide bonds in a “Y” configuration. The amino acid sequence runsfrom the N-terminal end at the top of the Y to the C-terminal end at thebottom of each chain. At the N-terminal end is a variable region (ofapproximately 100 amino acids in length) that provides for thespecificity of antigen binding.

The present invention provides improved methods for production ofimmunoglobulins of all types, including, but not limited to full lengthantibodies and antibody fragments having a native sequence (i.e. thatsequence produced in response to stimulation by an antigen), singlechain antibodies which combine the antigen binding variable region ofboth the heavy and light chains in a single stably-folded polypeptidechain; univalent antibodies (which comprise a heavy chain/light chaindimer bound to the Fc region of a second heavy chain); “Fab fragments”which include the full “Y” region of the immunoglobulin molecule, i.e.,the branches of the “Y”, either the light chain or heavy chain alone, orportions, thereof (i.e., aggregates of one heavy and one light chain,commonly known as Fab'); “hybrid immunoglobulins” which have specificityfor two or more different antigens (e.g., quadromas or bispecificantibodies as described for example in U.S. Pat. No. 6,623,940);“composite immunoglobulins” wherein the heavy and light chains mimicthose from different species or specificities; and “chimeric antibodies”wherein portions of each of the amino acid sequences of the heavy andlight chain are derived from more than one species (i.e., the variableregion is derived from one source such as a murine antibody, while theconstant region is derived from another, such as a human antibody).

The compositions and methods of the invention find utility in productionof immunoglobulins or fragments thereof wherein the heavy or light chainis “mammalian”, “chimeric” or modified in a manner to enhance itsefficacy. Modified antibodies include both amino acid and nucleic acidsequence variants which retain the same biological activity of theunmodified form and those which are modified such that the activity isaltered, i.e., changes in the constant region that improve complementfixation, interaction with membranes, and other effector functions, orchanges in the variable region that improve antigen bindingcharacteristics. The compositions and methods of the invention furtherinclude catalytic immunoglobulins or fragments thereof.

A “variant” immunoglobulin-encoding polynucleotide sequence may encode a“variant” immunoglobulin amino acid sequence that is altered by one ormore amino acids from the reference polypeptide sequence. The variantpolynucleotide sequence may encode a variant amino acid sequence thatcontains “conservative” substitutions, wherein the substituted aminoacid has structural or chemical properties similar to the amino acidwhich it replaces. In addition, or alternatively, the variantpolynucleotide sequence may encode a variant amino acid sequence thatcontains “non-conservative” substitutions, wherein the substituted aminoacid has dissimilar structural or chemical properties to the amino acidthat it replaces. Variant immunoglobulin-encoding polynucleotides mayalso encode variant amino acid sequences that contain amino acidinsertions or deletions, or both.

The present invention contemplates immunoglobulin sequence variantswhich encode biologically active immunoglobulins or fragments thereof,wherein the immunoglobulin polypeptide sequence or the nucleotidesequence encoding it has 80, 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99% or more sequence identity to the native sequence.

Furthermore, a variant “immunoglobulin-encoding polynucleotide” mayencode the same polypeptide as the reference polynucleotide sequencebut, due to the degeneracy of the genetic code, has a polynucleotidesequence altered by one or more bases from the reference polynucleotidesequence. Immunoglobulin sequence variants that encode a polypeptidewith the same biological activity as the immunoglobulin polypeptidesdescribed herein and hybridize under moderate to high stringencyhybridization conditions are considered to be within the scope of thepresent invention.

The term “fragment,” when referring to a recombinant immunoglobulin ofthe invention means a polypeptide which has an amino acid sequence whichis the same as part of but not all of the amino acid sequence of thecorresponding full length immunoglobulin protein, which either retainsessentially the same biological function or activity as thecorresponding full length protein, or retains at least one of thefunctions or activities of the corresponding full length protein. Thefragment preferably includes at least 20-100, 20-150 or 20-200contiguous amino acid residues of the full-length immunoglobulin.

The potential of antibodies as therapeutic modalities is currentlylimited by long time frame needed to select clones that producecommercially practical levels of immunoglobulin, the production capacityand excessive cost of the current technology. An improved v expressionsystem for immunoblobulin production would permit the expression anddelivery of two or more coding sequences, i.e., immunoglobulins with bi-or multiple-specificities from a single vector. The present inventionaddresses these limitations and is applicable to any immunoglobulin(i.e. an antibody) or fragment thereof as further detailed herein,including engineered antibodies such as single chain antibodies,full-length antibodies or antibody fragments.

Antibody Production

In one example of the present invention, the coding sequence for a firstor second chain of a protein or polypeptide is the coding sequence forthe heavy chain or a fragment thereof for any immunoglobulin, e.g., IgG,IgM, IgD, IgE or IgA. Alternatively, the coding sequence for a first orsecond chain of a protein or polypeptide is the coding sequence for thelight chain or a fragment thereof for an IgG, IgM, IgD, IgE or IgA.Genes for whole antibody molecules as well as modified or derived formsthereof, such as fragments, e.g., Fab, single chain Fv(scFv) and F(ab')₂are included within the scope of the invention. The antibodies andfragments can be animal-derived, human-mouse chimeric, humanized,Delmmunized™ or fully human. The antibodies can be bispecific andinclude but are not limited to diabodies, quadroma, mini-antibodies,ScBs antibodies and knobs-into-holes antibodies.

In practicing the invention, the production of an antibody, or variant(analogue) or fragment thereof using recombinant DNA technology can beachieved by culturing a modified recombinant host cell under cultureconditions appropriate for the growth of that host cell resulting inexpression of the coding sequences. In order to monitor the success ofexpression, antibody levels with respect to the antigen may be monitoredusing standard techniques such as ELISA, RIA, Western blot and the like.The antibodies are recovered from the culture supernatant using standardtechniques known in the art. Purified forms of these antibodies can, ofcourse, be readily prepared by standard purification techniques, e.g.,affinity chromatography via protein A, protein G or protein L columns,or based on binding to the particular antigen, or the particular epitopeof the antigen for which specificity is desired. Antibodies can also bepurified with conventional chromatography, such as an ion exchange orsize exclusion column, in conjunction with other technologies, such asammonia sulfate precipitation and size-limited membrane filtration.Preferred expression systems are designed to include signal peptides sothat the resulting antibodies are secreted into the culture medium orsupernatant, allowing for ease of purification, however, intracellularproduction is also possible.

The production and selection of antigen-specific fully human monoclonalantibodies from mice engineered with human Ig loci, has previously beendescribed (Jakobovits A. et al., Advanced Drug Delivery Reviews Vol. 31,pp: 33-42 (1998); Mendez M, et al., Nature Genetics Vol. 15, pp: 146-156(1997); Jakobovits A. et al., Current Opinion in Biotechnology Vol. 6,No. 5, pp: 561-566 (1995); Green L, et al., Nature Genetics Vol. 7, No.1, pp: 13-21(1994).

The production and recovery of the antibodies themselves can be achievedin various ways known in the art (Harlow et al., Antibodies, ALaboratory Manual, Cold Spring Harbor Lab, 1988).

Protein Coding Sequences

As used herein, a “first protein coding sequence” refers to aheterologous nucleic acid sequence encoding a polypeptide or proteinmolecule or domain or chain thereof including, but not limited to achain of an antibody or immunoglobulin molecule or fragment thereof, acytokine or fragment thereof, a growth factor or fragment thereof, achain of a Factor VIII molecule, a soluble or membrane-associatedreceptor or fragment thereof, a viral protein or fragment thereof, animmunogenic protein or fragment thereof, a transcriptional regulator orfragment thereof, a proapoptotic molecule or fragment thereof, a tumorsuppressor or fragment thereof, an angiogenesis inhibitor or fragmentthereof, etc.

As used herein, a “second protein coding sequence” refers to aheterologous nucleic acid sequence encoding: a polypeptide or proteinmolecule or domain or chain thereof including, but not limited to achain of an antibody or immunoglobulin or fragment thereof, a cytokineor fragment thereof, a growth factor or fragment thereof, a chain of aFactor VIII molecule, a soluble or membrane-associated receptor orfragment thereof, a viral protein or fragment thereof, an immunogenicprotein or fragment thereof, a transcriptional regulator or fragmentthereof, a proapoptotic molecule or fragment thereof, a tumor suppressoror fragment thereof, an angiogenesis inhibitor or fragment thereof, etc.

The lentivector constructs of the invention may comprise two or moretransgenes or heterologous coding sequences, e.g., a first proteincoding sequence, a second protein coding sequence, a third proteincoding sequence, etc. The two or more transgenes may be delivered to acell using one or more lentivectors. When a single lentivector isemployed the two or more transgenes are co-expressed by operativelinkage to a single promoter and a self processing cleavage sequencesuch as 2A. Numerous transgenes may be employed in the practice of thepresent invention and include, but are not limited to, nucleotidesequences encoding one or more of the proteins indicated below or afragment thereof;

1. A sequence encoding HIF-1α and HIFβ (HIF), p35 and p40 (IL-12), chainA and chain B of insulin, integrins such as, but not limited to alpha Vbeta 3 or alpha V beta 5, antibody heavy and light chains and the heavyand light chain of Factor VIII.

2. A sequence encoding a soluble receptor, include but are not limitedto, the TNF p55 and p75 receptor, the IL-2 receptor, the FGF receptors,the VEGF receptors, TIE2, the IL-6 receptor and the IL-1 receptor;

3. A sequence encoding a cytokine including, but not limited to, anyknown or later discovered cytokine, for example, IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL10, IL-11, IL-12, IL-13, IL-18, IL-24,INF-α, INF-β, INF-γ, GM-CSF, G-CSF and erythropoietin.

4. A sequence encoding a growth factor including, but not limited to,VEGF, FGF, Angiopoietin-1 and 2, PDGF, EGF, IGF, NGF, IDF, HGF, TGF-α,TGF-beta.

5. A sequence encoding a pro-apoptotic factor including, but not limitedto, Bad, Bak, Bax, Bcl2, Bcl-Xs, Bik, Caspases, FasL, and TRAIL.

6. A sequence encoding a tumor suppressor protein or cell cycleregulator including, but not limited to, p53, p16, p19,-21, p27, PTEN,RB1.

7. A sequence encoding an angiogenesis regulator including, but notlimited to, angiostatin, endostatin, TIMPs, antithrombin, plateletfactor 4 (PF4), soluble forms of VEGFR1 (domains 1-7) and VEGFR2(domains 1-7) fused to an Fc segment of IgG1, VEGF-TRAP, PEDF, PEX,troponin I, thrombospondin, tumstatin, 16 Kd Prolactin.

Cloned sequences and full-length nucleotides encoding any of theabove-referenced biologically active molecules may be obtained bywell-known methods in the art (Sambrook et al., 1989). In general, thenucleic acid coding sequences are known and may be obtained from publicdatabases and/or scientific publications.

Homologues and variants of heterologous protein and polypeptide codingsequences are included within the scope of the invention based on“sequence identity” or “% homology” to known nucleic acid sequenceswhich are available in public databases and/or selective hybridizationunder stringent conditions to such known nucleic acid sequences (asdescribed above for self processing cleavage sequences). Homologues andvariants of heterologous protein and polypeptide amino acid sequencesand nucleic acid sequences that encode them are further included withinthe scope of the invention. Such sequences may be identified based on“sequence identity” to known sequences using publicly availabledatabases and sequence alignment programs, as described above forself-processing cleavage sequences.

The present invention contemplates heterologous protein and polypeptidevariants which encode biologically active proteins, polypeptides orfragments thereof, wherein the protein or polypeptide sequence or thenucleotide sequence encoding it has 80, 85, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99% or more sequence identity to the native sequence.

Furthermore, a variant “heterologous protein or polypeptide-encodingpolynucleotide” may encode the same polypeptide as the referencepolynucleotide sequence but, due to the degeneracy of the genetic code,has a polynucleotide sequence altered by one or more bases from thereference polynucleotide sequence. Heterologous protein and polypeptidesequence variants that encode a polypeptide with the same biologicalactivity as heterologous protein or polypeptide described herein andhybridize under moderate to high stringency hybridization conditions areconsidered to be within the scope of the present invention.

Furthermore, a variant “immunoglobulin-encoding polynucleotide” mayencode the same polypeptide as the reference polynucleotide sequencebut, due to the degeneracy of the genetic code, has a polynucleotidesequence altered by one or more bases from the reference polynucleotidesequence. Immunoglobulin sequence variants that encode a polypeptidewith the same biological activity as the immunoglobulin polypeptidesdescribed herein and hybridize under moderate to high stringencyhybridization conditions are considered to be within the scope of thepresent invention.

Protein Expression

It will be understood that the lentivectors of the invention findutility in the expression of recombinant proteins and polypeptides inany lentiviral-based protein expression system, a number of which areknown in the art and examples of which are described herein.

Following expression, recombinant proteins are recovered from theculture using standard techniques known in the art. The production andrecovery of recombinant proteins themselves can be achieved in variousways numerous examples of which are known in the art. For example, theproduction of a recombinant protein, polypeptide, an analogue orfragment thereof, can be undertaken by culturing the modifiedrecombinant host cells under culture conditions appropriate that hostcell resulting in expression of the coding sequence(s). In order tomonitor the success of expression, recombinant protein or polypeptidelevels are monitored using standard techniques such as ELISA, RIA,Western blot and the like.

Purified forms of the recombinant proteins can, of course, be readilyprepared by standard purification techniques known in the art, e.g.,affinity chromatography. Recombinant proteins can also be purified usingconventional chromatography, such as an ion exchange or size exclusioncolumn, in conjunction with other technologies, such as size-limitedmembrane filtration. The expression systems are preferably designed toinclude signal peptides so that the resulting recombinant proteins aresecreted into the medium, however, intracellular production is alsopossible.

The operability of the present invention has been demonstrated byexpression of immunoglobulin heavy and light chains using theself-processing cleavage sequence-containing lentivectors of the presentinvention (See, e.g., Example 2). The advantages associated with use ofself-processing cleavage sequences are enhanced by inclusion of anadditional proteolytic cleavage site between the coding sequence for afirst protein or polypeptide and the self-processing cleavage sequencein the vectors of the invention, resulting in removal of amino acidresidues associated with the self-processing cleavage sequence.Efficient removal of 2A residues by incorporation of a furin cleavagesite in the vectors of the invention is demonstrated U.S. PatentPublication Nos. 20040265955 and 20050003482.

Vectors for use in Practicing the Invention

Retroviral vectors are also a common tool for gene delivery (Miller,Nature 357: 455-460, 1992). Retroviral vectors and more particularlylentiviral vectors may be used in practicing the present invention.Accordingly, the term “retrovirus” or “retroviral vector”, as usedherein is meant to include “lentivirus” and “lentiviral vectors”respectively. Retroviral vectors have been tested and found to besuitable delivery vehicles for the stable introduction of genes ofinterest into the genome of a broad range of target cells. The abilityof retroviral vectors to deliver unrearranged, single copy transgenesinto cells makes retroviral vectors well suited for transferring genesinto cells. Further, retroviruses enter host cells by the binding ofretroviral envelope glycoproteins to specific cell surface receptors onthe host cells. Consequently, pseudotyped retroviral vectors in whichthe encoded native envelope protein is replaced by a heterologousenvelope protein that has a different cellular specificity than thenative envelope protein (e.g., binds to a different cell-surfacereceptor as compared to the native envelope protein) may also findutility in practicing the present invention. The ability to direct thedelivery of retroviral vectors encoding one or more target proteincoding sequences to specific target cells is desirable in practice ofthe present invention.

The invention relates to retroviral vectors, producer cells, andproducer cell lines. In particular, the invention relates to a novelapproach for the expression of multimeric, heterologous coding sequencesin a single mammalian cell using one or more self-inactivating (“SIN”)retroviral vectors that encode a heterologous sequence. Moreparticularly, the retroviral vectors are SIN lentiviral vectors. Theinvention further relates to methods of using SIN lentiviral vectors formaking multimeric recombinant proteins.

The present invention provides retroviral vectors that include e.g.,retroviral transfer vectors comprising one or more transgene sequencesand retroviral packaging vectors comprising one or more packagingelements. In particular, the present invention provides pseudotypedretroviral vectors encoding a heterologous or functionally modifiedenvelope protein for producing pseudotyped retrovirus. Preferably, theheterologous env gene comprises a VSV-G or baculoviral gp64 env gene,although those skilled in the art will appreciate that other env genesmay be employed.

One preferred method of vector production is transient transfection ofplasmids containing the viral packaging genes and the transgene into acell line. Alternatively, the vectors are produced via transfection,transduction or infection into a packaging cell line to make producercells. Methods for transfection, transduction or infection are wellknown by those of skill in the art. Both transiently transfected andproducer cells are effective to generate viral particles that containthe transgene. For either the stable or transient production method therecombinant virus is recovered from the culture media, concentratedand/or purified, and titrated by standard methods used by those of skillin the art.

The core sequence of the retroviral vectors of the present invention maybe readily derived from a wide variety of retroviruses, including forexample, B, C, and D type retroviruses as well as spumaviruses andlentiviruses (RNA Tumor Viruses, Second Edition, Cold Spring HarborLaboratory, 1985). An example of a retrovirus suitable for use in thecompositions and methods of the present invention includes, but is notlimited to, a lentivirus. Other retroviruses suitable for use in thecompositions and methods of the present invention include, but are notlimited to, Avian Leukosis Virus, Bovine Leukemia Virus, Murine LeukemiaVirus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus,Reticuloendotheliosis virus and Rous Sarcoma Virus. Preferred MurineLeukemia Viruses include 4070A and 1504A (Hartley and Rowe, J. Virol.19:19-25, 1976), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245),Graffi, Gross (ATCC No. VR-590), Kirsten, Harvey Sarcoma Virus andRauscher (ATCC No. VR-998), and Moloney Murine Leukemia Virus (ATCC No.VR-190). Such retroviruses may be readily obtained from depositories orcollections such as the American Type Culture Collection (“ATCC”;Rockville, Md.), or isolated from known sources using commonly availabletechniques.

Preferably, a retroviral vector sequence of the present invention isderived from a lentivirus. A preferred lentivirus is a humanimmunodeficiency virus, e.g., type 1 or 2 (i.e., HIV-1 or HIV-2, whereinHIV-1 was formerly called lymphadenopathy associated virus 3 (HTLV-III)and acquired immune deficiency syndrome (AIDS)-related virus (ARV)), oranother virus related to HIV-1 or HIV-2 that has been identified andassociated with AIDS or AIDS-like disease. Other lentiviruses include asheep Visna/maedi virus, a feline immunodeficiency virus (FIV), a bovinelentivirus, simian immunodeficiency virus (SIV), an equine infectiousanemia virus (EIAV), and a caprine arthritis-encephalitis virus (CAEV).

The various genera and strains of retroviruses suitable for use in thecompositions and methods are well known in the art (see, e.g., FieldsVirology, Third Edition, edited by B. N. Fields et al., Lippincott-RavenPublishers (1996), see e.g., Chapter 58, “Retroviridae: The Viruses andTheir Replication, Classification”, pages 1768-1771, including Table 1.

The packaging system used to generate retroviral vectors is composed ofat least two packaging vectors, a first packaging vector which comprisesa first nucleotide sequence comprising a gag, a pol, or gag and polgenes and a second packaging vector which comprises a second nucleotidesequence comprising a heterologous or functionally modified envelopegene. In a preferred embodiment, the retroviral elements are derivedfrom a lentivirus, such as HIV. Preferably, the vectors lack afunctional tat gene and/or functional accessory genes (vif, vpr, vpu,vpx, nef). In a further preferred embodiment, the system furthercomprises a third packaging vector that comprises a nucleotide sequencecomprising a rev gene. The packaging system can be provided in the formof a packaging cell that contains the first, second, and, optionally,third nucleotide sequences.

The invention is applicable to a variety of systems, and those skilledin the art will appreciate the common elements shared across differinggroups of retroviruses. The description herein uses lentiviral systemsas a representative example. However, all retroviruses share thefeatures of enveloped virions with surface projections and containingone molecule of linear, positive-sense single stranded RNA, a genomeconsisting of a dimer, and the common proteins gag, pol and env.

Lentiviruses share several structural virion proteins in common,including the envelope glycoproteins SU (gp120) and TM (gp4l), which areencoded by the env gene; CA (p24), MA (p17) and NC (p7-11), which areencoded by the gag gene; and RT, PR and IN encoded by the pol gene.HIV-1 and HIV-2 contain accessory and other proteins involved inregulation of synthesis and processing virus RNA and other replicativefunctions. The accessory proteins, encoded by the vif, vpr, vpu, vpx,and nef genes, can be omitted (or inactivated) from the recombinantsystem. In addition, tat and rev can be omitted or inactivated, e.g., bymutation or deletion.

First generation lentiviral vector packaging systems provide separatepackaging constructs for gag/pol and env, and typically employ aheterologous or functionally modified envelope protein for safetyreasons. See e.g., Miller and Buttimore, Molec. Cell. Biol. 6(8):2895-2902 (1986). These modifications minimize the homology between thepackaging genome and the viral vector so that the ability of the vectorto form recombinants is reduced (see e.g., Miller and Rosman,BioTechniques 7(9):980-990 (1989)).

In second generation lentiviral vector systems, the accessory genes,vif, vpr, vpu and nef, are deleted or inactivated and the packagingfunctions are divided into two genomes: one genome expresses the gag andpol gene products, and the other genome expresses the env gene product(see e.g., Bosselman et al., Molec. Cell. Biol. 7(5):1797-1806 (1987);Markowitz et al., J. Virol. 62(4):1120-1124 (1988); Danos and Mulligan,Proc. Nat'l. Acad. Sci. (USA) 85:6460-6464 (1988)). This approacheliminates the ability for co-packaging and subsequent transfer of thepsi-genome (containing the viral packaging element psi), as well assignificantly decreases the frequency of recombination due to thepresence of three retroviral genomes in the packaging cell that mustundergo recombination to produce RCR. In the event recombinants arise,mutations or deletions within the undesired gene products renderrecombinants non-functional (see e.g., Danos and Mulligan, supra Danosand Mulligan, supra; Boselman et al., supra; and Markowitz et al.,supra). In addition, the deletion of the 3′ LTR on both packagingfunction constructs further reduces the ability to form functionalrecombinants.

Third generation lentiviral vector systems are preferred for use inpracticing the present invention and include those from which the tatgene has been deleted or otherwise inactivated (e.g., via mutation).Compensation for the regulation of transcription normally provided bytat can be provided by the use of a strong constitutive promoter, suchas the human cytomegalovirus immediate early (HCMV-IE)enhancer/promoter. Other promoters/enhancers can be selected based onstrength of constitutive promoter activity, specificity for targettissue (e.g., a liver-specific promoter), or other factors relating todesired control over expression, as is understood in the art. Forexample, in some embodiments, it is desirable to employ an induciblepromoter such as tet to achieve controlled expression. The gene encodingrev is preferably provided on a separate expression construct, such thata typical third generation lentiviral vector system will involve fourplasmids: one each for gagpol, rev, envelope and the transfer vector.Regardless of the generation of packaging system employed, gag and polcan be provided on a single construct or on separate constructs.

Typically, the packaging vectors are included in a packaging cell, andare introduced into the cell via transfection, transduction orinfection. Methods for transfection, transduction or infection are wellknown by those of skill in the art. A retroviral/lentiviral transfervector of the present invention can be introduced into a packaging cellline, via transfection, transduction or infection, to generate aproducer cell or cell line. The packaging vectors of the presentinvention can be introduced into human cells or cell lines by standardmethods including, e.g., calcium phosphate transfection, lipofection orelectroporation. In some embodiments, the packaging vectors areintroduced into the cells together with a dominant selectable marker,such as neo, DHFR, Gln synthetase or ADA, followed by selection in thepresence of the appropriate drug and isolation of clones. A selectablemarker gene can be linked physically to genes encoding by the packagingvector.

Stable cell lines, wherein the packaging functions are configured to beexpressed by a suitable packaging cell, are known. For example, see U.S.Pat. No. 5,686,279; and Ory et al., Proc. Natl. Acad. Sci. (1996)93:11400-11406, which describe packaging cells. Further description ofstable cell line production can be found in Dull et al., 1998, J.Virology 72(11):8463-8471; and in Zufferey et al., 1998, J. Virology72(12):9873-9880.

Zufferey et al., 1997, Nature Biotechnology 15:871-875, teach alentiviral packaging plasmid wherein sequences 3′ of pol including theHIV-1 envelope gene are deleted. The construct contains tat and revsequences and the 3′ LTR is replaced with poly A sequences. The 5′ LTRand psi sequences are replaced by another promoter, such as one that isinducible. For example, a CMV promoter or derivative thereof can beused.

Preferred packaging vectors may contain additional changes to thepackaging functions to enhance lentiviral protein expression and toenhance safety. For example, all of the HIV sequences upstream of gagcan be removed. Also, sequences downstream of the envelope can beremoved. Moreover, steps can be taken to modify the vector to enhancethe splicing and translation of the RNA.

Optionally, a conditional packaging system is used, such as thatdescribed by Dull et al., J. Virology 72(11):8463-8471, 1998. Alsopreferred is the use of a self-inactivating vector (SIN), which improvesthe biosafety of the vector by deletion of the HIV-1 long terminalrepeat (LTR) as described, for example, by Zufferey et al., 1998, J.Virology 72(12):9873-9880. Inducible vectors can also be used, such asthrough a tet-inducible LTR.

Any vector for use in practicing the invention will include heterologouscontrol sequences, such as a constitutive promoter, e.g., thecytomegalovirus (CMV) immediate early promoter, the RSV LTR, the MoMLVLTR, and the PGK promoter; tissue or cell type specific promotersincluding mTTR, TK, HBV, hAAT, regulatable or inducible promoters,enhancers, etc. Preferred promoters include the LSP promoter (Ill etal., Blood Coagul. Fibrinolysis 852:23-30, 1997), the EF1-alpha promoter(Kim et al., Gene 91(2):217-23, 1990) and Guo et al., Gene Ther.3(9):802-10, 1996). Most preferred promoters include the elongationfactor 1-alpha (EF1a) promoter, a phosphoglycerate kinase-1 (PGK)promoter, a cytomegalovirus immediate early gene (CMV) promoter,chimeric liver-specific promoters (LSPs), a cytomegalovirusenhancer/chicken beta-actin (CAG) promoter, a tetracycline responsivepromoter (TRE), a transthyretin promoter (TTR), a MND promoter, a simianvirus 40 (SV40) promoter and a CK6 promoter. Other promoters/enhancerscan be selected based on strength of constitutive promoter activity,specificity for target tissue or other factors relating to desiredcontrol over expression, as is understood in the art. The sequences ofthese and numerous additional promoters are known in the art. Therelevant sequences may be readily obtained from public databases andincorporated into vectors for use in practicing the present invention.

The invention uses lentiviral vectors, particles, packaging systems andproducer cells capable of producing a high titer recombinant lentiviruscapable of selectively infecting human and other mammalian cells. In oneembodiment, the recombinant lentivirus of the invention has a titer ofgreater than 5×10⁵ infectious units/ml. Preferably, the recombinantretrovirus has a titer of greater than 1×10⁶ infectious units/ml.Typically, titer is determined by conventional infectivity assay on293T, HeLa or HUH7 hepatoma cells.

The present invention also contemplates the inclusion of a generegulation system for the controlled expression of the coding sequencefor two or more polypeptides or proteins of interest. Gene regulationsystems are useful in the modulated expression of a particular gene orgenes. In one exemplary approach, a gene regulation system or switchincludes a chimeric transcription factor that has a ligand bindingdomain, a transcriptional activation domain and a DNA binding domain.The domains may be obtained from virtually any source and may becombined in any of a number of ways to obtain a novel protein. Aregulatable gene system also includes a DNA response element thatinteracts with the chimeric transcription factor. This element islocated adjacent to the gene to be regulated.

Exemplary gene regulation systems that may be employed in practicing thepresent invention include, the Drosophila ecdysone system (Yao et al.,Proc. Nat. Acad. Sci., 93:3346 (1996)), the Bombyx ecdysone system (Suhret al., Proc. Nat. Acad. Sci., 95:7999 (1998)), the Valentis GeneSwitch®synthetic progesterone receptor system which employs RU-486 as theinducer (Osterwalder et al., Proc Natl Acad Sci 98(22):12596-601(2001)); the Tet™ & RevTet™ Systems (BD Biosciences Clontech), whichemploys small molecules, such as tetracycline (Tc) or analogues, e.g.doxycycline, to regulate (turn on or off) transcription of the target(Knott et al., Biotechniques 32(4):796, 798, 800 (2002)); ARIADRegulation Technology which is based on the use of a small molecule tobring together two intracellular molecules, each of which is linked toeither a transcriptional activator or a DNA binding protein. When thesecomponents come together, transcription of the gene of interest isactivated. Ariad has two major systems: a system based onhomodimerization and a system based on heterodimerization (Rivera etal., Nature Med, 2(9):1028-1032 (1996); Ye et al., Science 283: 88-91(2000)), either of which may be incorporated into the vectors of thepresent invention.

Preferred gene regulation systems for use in practicing the presentinvention are the ARIAD Regulation Technology and the Tet™ & RevTet™Systems.

Delivery Of Nucleic Acid Constructs Including Protein Or PolypeptideCoding Sequences To Cells

The vector constructs of the invention comprising nucleic acid sequencesencoding heterologous proteins or polypeptides, and a self-processingcleavage site alone or in combination with a sequence encoding anadditional proteolytic cleavage site may be introduced into cells invitro, ex vivo or in vivo for expression of heterologous codingsequences by cells, e.g., somatic cells in vivo, or for the productionof recombinant polypeptides by vector-transduced cells, in vitro or invivo.

The vector constructs of the invention may be introduced into cells invitro or ex vivo using standard methodology known in the art. Suchtechniques include transfection using calcium phosphate, microinjectioninto cultured cells (Capecchi, Cell 22:479-488 (1980)), electroporation(Shigekawa et al., BioTechn., 6:742-751 (1988)), liposome-mediated genetransfer (Mannino et al., BioTechn., 6:682-690 (1988)), lipid-mediatedtransduction (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417(1987)), and nucleic acid delivery using high-velocity microprojectiles(Klein et al., Nature 327:70-73 (1987)).

For in vitro or ex vivo expression, any cell capable of expressing afunctional protein may be employed. Numerous examples of cells and celllines used for protein expression are known in the art. For example,prokaryotic cells and insect cells may be used for expression. Inaddition, eukaryotic microorganisms, such as yeast may be used. Theexpression of recombinant proteins in prokaryotic, insect and yeastsystems are generally known in the art and may be adapted for protein orpolypeptide expression using the compositions and methods of the presentinvention.

Exemplary host cells useful for expression further include mammaliancells, such as fibroblast cells, cells from non-human mammals such asovine, porcine, murine and bovine cells, insect cells and the like.Specific examples of mammalian cells include COS cells, VERO cells, HeLacells, Chinese hamster ovary (CHO) cells, 293 cell, NSO cells, 3T3fibroblast cells, W138 cells, BHK cells, HEPG2 cells, DUX cells and MDCKcells.

Host cells are cultured in conventional nutrient media, modified asappropriate for inducing promoters, selecting transformants, oramplifying the genes encoding the desired sequences. Mammalian hostcells may be cultured in a variety of media. Commercially availablemedia such as Ham's F10 (Sigma), Minimal Essential Medium (MEM), Sigma),RPMI 1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma)are typically suitable for culturing host cells. A given medium isgenerally supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferring, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleosides (such as adenosine and thymidine),antibiotics, trace elements, and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theappropriate culture conditions for a particular cell line, such astemperature, pH and the like, are generally known in the art, withsuggested culture conditions for culture of numerous cell lines forexample in the ATCC Catalogue available on line at“www.atcc.org/SearchCatalogs/AllCollections.cfm”

The lentivectors of the invention may also be administered in vivo viavarious routes (e.g., intradermally, intravenously, intratumorally, intothe brain, intraportally, intraperitoneally, intramuscularly, into thebladder, etc.), to deliver multiple genes to express two or moreproteins or polypeptides in animal models or human subjects. Dependentupon the route of administration, the therapeutic proteins elicit theireffect locally (e.g., in brain or bladder) or systemically (other routesof administration). The use of tissue specific promoters 5′ to the openreading frame(s) for a protein or polypeptide in the vectors of theinvention may be used to effect tissue specific expression of the two ormore proteins or polypeptides encoded by the vector.

Various methods that introduce recombinant lentivectors into targetcells in vitro, ex vivo or in vivo have been previously described andare well known in the art. For example, in vivo delivery of therecombinant vectors of the invention may be targeted to a wide varietyof organ types including, but not limited to brain, liver, bloodvessels, muscle, heart, lung and skin. In the case of ex vivo genetransfer, the target cells are removed from the host and geneticallymodified in the laboratory using recombinant vectors of the presentinvention and methods well known in the art.

The recombinant vectors of the invention can be administered usingconventional modes of administration including but not limited to themodes described above. The recombinant vectors of the invention may beprovided in any of a variety of formulations such as liquid solutionsand suspensions, microvesicles, liposomes and injectable or infusiblesolutions. The preferred form depends upon the mode of administrationand the therapeutic application. A from appropriate to the route ofdelivery may be readily determined using knowledge generally availableto those of skill in the relevant art.

Current methods of recombinant immunoglobulin production rely on use of:CHO cells, derivatives thereof; NSO cells; PerC.6 cells; and HEK cells.Most currently used methods for recombinant immunoglobulin productionare based on use of vector constructs which include an amplifiable genesuch as dihydrofolate reductase (DHFR). As a result the process ofrecombinant protein production requires the steps of: (1) transfection;(2) selection by culture in medium containing a drug such as Neomycin;(3) amplification of the immunoglobulin coding sequence based on thepresence of DHFR in the construct and culture in medium containing anear lethal concentration of methotrexate; (4) screening for viablecells; and (5) further step-wise amplification in culture mediumcontaining increasing concentrations of methotrexate; and (6) furtherscreening at each amplification step to identify viable cells.

Using the DHFR system, the genomic copy number of the nucleic acidencoding the immunoglobulin is increased by the selective pressure ofexposing cells to methotrexate, a drug that blocks the activity of DHFR,resulting in higher levels of antibody expression. After 2 to 3 weeks ofexposure to methotrexate at a near lethal concentration, the majority ofcells die, but cells that overproduce DHFR will survive. Multiple roundsof step-wise amplification in methotrexate-containing medium aretypically required, which typically takes from 6 to 12 months, to selecta clone that produces at least 20 pg/c/day (pg per cell per day).Frequently as many as 2000-4000 initial clones are screened in order toselect a clone that produces at least 20 pg/c/day.

The protein expression levels of different cell clones obtained fromstep-wise methotrexate amplification can vary widely. As a consequence,the identification of high-producer cell lines is a tedious andlabor-intensive process. Several methods for the isolation of clonesexist, the most popular being limiting dilution cloning.

In contrast, the methods of the current invention do not requireamplification methotrexate-containing medium and hence multiple roundsof successive culturing and screening are avoided. The efficiency ofinfection and ability of the lentiviral vector to re-infect cellsmultiple times allow for the rapid generation of cell lines containingnumerous genomic copies of the nucleic acid encoding the antibody 2Afusion protein. As a result, 10-fold to 50-fold fewer clones are neededfor screening to select a clone that produces at least 20 pg/c/day,shortening the clonal selection process by as much as about 10 months.

For products, like monoclonal antibodies, cell lines must produce atleast 20 pg/cell/day to be suitable candidates for commercialproduction. The combination of fast growth and high productivity makes acell line a candidate for commercial production. A further importantconsideration is the stability of the cell line over extended periods oftime and upon scale up.

Some clonal cell lines, such as GS-NS0 have been found to be unstableafter long term culture. In addition, the presence of methotrexate inlong term culture has been shown to result in undesirable geneticheterogeneity in the cells.

The present invention provides advantages in that: 1) the process doesnot rely on inclusion of an amplifiable gene such as dihydrofolatereductase (DHFR) in the expression construct or use of an agent such asmethotrexate for amplification; 2) the process is less expensive becauseof the fact that the resulting cell line need not be methotrexateresistant thereby eliminating the need to add exogenous methotrexate tothe culture medium and allows for additional cell lines capable of largescale culture to be employed in the methods of the invention; 3) thescreening time is significantly reduced because the process requiresless steps than current commercial processes. The process only requirestransfection and selection in a medium containing a drug such asneomycin. No amplification or subsequent rounds of screening arerequired; 4) the cell line is preferably transfected or infectedmultiple times over a short period of time to rapidly increase thegenomic copy number of the vector. The number of transfections orinfections can vary depending, in part, on the coding sequence of theantibody to be expressed, strength of the selected promoter and parentcell line used for expression. In certain embodiments, at least 3 roundsof transfection or infection (“pings”) are employed to get optimalexpression of a heterologous coding sequence by way of a lentivirusvector; 5) the time for selection of high producer clones isdramatically reduced from the typical 6-12 months to 1-2 months; and 6)the instability of the producer cell line over extended periods of timeand upon scale up is less likely to be a problem due to the absence ofmethotrexate in the culture medium.

In one preferred embodiment, for immunoglobulin production, clonal celllines of the invention produce at least 20 pg/cell/day, preferably atleast 25, 30, 35, 40, 45 or 50, 60, 70, 80, 90, 100, 125, 150 or 200pg/cell/day. In another preferred embodiment, the timing for selectionof clones that produce at least 20 pg/cell/day is less than 4 months,preferably less than 3 months and more preferably less than 2 months. Inyet another preferred embodiment temperature for culture is at least31°.

In one preferred embodiment, for immunoglobulin production, clonal celllines of the invention comprise at least 20, preferably at least 25, 30,35, 40, 45 or 50, 60, 70, 80, 90, 100 genomic copies of the lentiviralvector comprising the nucleic acid encoding the immunoglobulin 2Aconstruct.

In yet another preferred embodiment, clonal cell lines of the inventionproduce at least 20 pg/cell/day, preferably at least 25, 30, 35, 40, 45or 50, 60, 70, 80, 90, 100, 125, 150 or 200 pg/cell/day and comprise atleast 20 genomic copies of the lentiviral vector comprising the nucleicacid encoding the immunoglobulin 2A construct.

The many advantages of the invention to be realized in recombinantprotein and polypeptide production in vivo include administration of asingle vector for long-term and sustained expression of two or morerecombinant protein or polypeptide ORFs in patients; in vivo expressionof two or more recombinant protein or polypeptide ORFs having biologicalactivity; and the natural posttranslational modifications of therecombinant protein or polypeptide generated in human cells.

One preferred aspect is use of the recombinant vector constructs of thepresent invention for the in vitro production of recombinant proteinsand polypeptides. Methods for recombinant protein production are wellknown in the art and self processing cleavage site-containing vectorconstructs of the present invention may be utilized for expression ofrecombinant proteins and polypeptides using such standard methodology.

In one exemplary aspect of the invention, lentivector introduction oradministration to a cell is carried out by:

(1) introduction or administration of the lentivector to a cell by morethan one round of transfection/transduction or infection;

(2) culturing the infected cell under conditions that select for a cellexpressing the recombinant protein or polypeptide e.g., in mediumcontaining a selection agent such as Neomycin;

(3) evaluating expression of the recombinant protein or polypeptide; and

(4) collecting the recombinant protein or polypeptide.

In a preferred embodiment the cells are transfected or infected at least3 times, more preferably at least 4 or 5 times.

Methods and Compositions of the Invention

The invention relates to engineered lentiviral vectors for expression oftwo or more domains or chains of a multimeric protein. In one aspect themultimeric protein is an immunoglobulin and full-length antibody heavyand light chain coding sequences are expressed using a lentivectorcomprising a single open reading frame driven by a single promoterwherein the vector comprises a self-processing cleavage site or sequencebetween the heavy and light chain coding sequences. In another aspectthe protein is a multimeric heterologous protein and the full-lengthcoding sequences are expressed using a lentivector comprising a singleopen reading frame driven by a single promoter wherein the vectorcomprises one or more self-processing cleavage sites or sequences.

In yet another aspect, the invention provides a method for high levelexpression of recombinant protein using more than one engineeredlentivector, wherein each lentivector encodes a single open readingframe of a multimeric protein driven by a single promoter. For example,for expression of a full-length antibody, individual lentivectors thatencode the full-length antibody heavy and light chain, respectively, areused to infect the same cell such that high-level expression of abiologically active antibody results.

In one preferred embodiment, individual populations of host cells aretransduced with lentiviral transfer vectors wherein the heterologousprotein coding sequence encoded by the vectors is not the same andwherein each lentivector comprises the coding sequence for a singledomain or chain of a heterologous protein operably linked to anexpression control sequence. For example, a population of cells istransformed with a transfer vector comprising a heterologous proteincoding sequence, such as an immunoglobulin light chain coding sequenceoperably linked to an expression control sequence, and this populationor clones derived from it are transduced with a second transfer vectorcomprising a second heterologous protein coding sequence, such as animmunoglobulin heavy chain coding sequence operably linked to anexpression control sequence. The resulting population is cultured underconditions suitable for production of the multimeric protein.

In another preferred embodiment, the transfer vectors further comprisefirst a strong promoter (e.g. CMV, SV40, MND, or CAG), followed by theantibody heavy chain sequence (H), a furin cleavage site (F), a 2Aself-processing sequence derived from the Foot-and-Mouth Disease virus(2A), and an antibody light chain sequence (L). The resulting constructis designated H-F-2A-L. The 2A peptide sequence provides a “cleavage”site that facilitates the generation of two polypeptide chains of theantibody molecule during the translation process as shown in FIG. 1. Thefurin cleavage site provides a secondary cleavage during antibodysecretion pathway to remove 2A residues that are attached to the Cterminus of the first gene (i.e. heavy chain in this construct). In yetanother preferred embodiment, the transfer vectors further comprisefirst a strong promoter, followed by the antibody light chain sequence(L), a furin cleavage site (F), a 2A self-processing sequence derivedfrom the Foot-and-Mouth Disease virus (2A), and an antibody heavy chainsequence (H). The resulting construct is designated L-F-2A-H.

Recombinant lentiviral particles were generated and used to transducehuman or hamster cells in vitro. ELISA assays for antibody expressionfrom these supernatants revealed that the antibody was produced at highlevels in both 293 and CHO cells transduced with CAG H-F-2A-L lentiviralparticles. The ratio of the heavy and light chains expressed from theH-F-2A-L construct was approximately 1:1, and the final antibodyretained full biological activity based on antibody binding andneutralizing assays.

The present invention finds utility in expression of a full-lengthmonoclonal antibody (IgG, IgM, IgD, IgE, IgA) or antibody fragments frommammalian cells transduced with lentiviral vectors as well as expressionof any heterodimeric protein. Given the high transduction efficacy andgene expression level, lentiviral vectors are able to rapidly generatestable cells lines that express high levels of recombinant proteins suchas antibodies. In one preferred embodiment, the lentiviral vectorcomprises a self-processing site or sequence.

The objects of the invention have been achieved by a series ofexperiments, some of which are described by way of the followingnon-limiting examples.

Examples Example 1 Construction and Production of 2A Antibody ExpressionConstructs Transfection Plasmids

In order to generate lentivector constructs encoding a rat anti-mouseVEGFR2 and human anti-KDR antibody, DNA fragments that encode theantibody heavy chain, furin cleavage site, 2A sequence, and antibodylight chain were linked together by PCR extension. A DNA fragmentincluding a furin cleavage site RAKR (SEQ ID NO: 18), and the FMDV 2Asequence APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 6), were amplified from acloned plasmid by PCR. The heavy and light chain fragments wereamplified from the cloned plasmids that encode the full-length antibodyheavy or light chains respectively. During PCR, an EcoR I restrictionendonucleotidase site was added to the 5′ prime end of the heavy chainand the 3′ prime end of the light chain. The fused heavy chain-furincleavage site-2A-light chain DNA fragment was digested with EcoR I andpurified via agarose gel. The purified DNA fragment was inserted intothe pDHFR plasmid at the EcoR I site using T4 DNA ligase. The pDHFRcontains a CAG promoter operatively liked to the antibody codingsequences and an SV 40 promoter operatively liked to the DHFR gene. Anative signal peptide (leader) was included in the heavy or light chain,respectively, to facilitate secretion of the polypeptides uponsynthesis. In addition, the construct also contains a polyA sequence toensure high-level gene expression (FIG. 2). A pDHFR dual CAG Abexpression plasmid with a CAG promoter that drives the antibody lightchain and a second CAG promoter that drives the antibody heavy chain wasalso constructed (FIG. 2). This plasmid contains the same plasmidbackbone as pDHFR but encodes two CAG promoters. A multiple cloning siteand a polyA signal sequence follows each CAG promoter sequence. Togenerate the dual promoter antibody plasmid, an antibody heavy chain orlight chain coding sequence was amplified from a cloned plasmid thatcontains the antibody heavy or light chain sequence, respectively. Theantibody light chain sequence was inserted after the first CAG promoter,and the heavy chain after the second CAG promoter using the multiplecloning sites.

Lentiviral Plasmids: The nucleotide coding sequences encoding the KDRand DC101 HF2AL antibodies were cloned into 3^(rd) generation lentiviraltransfer vectors using standard molecular biology techniques routinelyemployed by those of skill in the art. The 3^(rd) generation lentiviralvector system has previously been described (Dull et al., J. Virol.72:8463-8471, 1998). Briefly, the transfer vector contains a 5′ chimericRSV/LTR promoter, cPPT (Zennou et al., Cell 101:173-185, 2000), CAGpromoter (Miyazaki et al., Gene 79:269-277, 1989), and SIN LTR (Zuffereyet al., J. Virol. 72:9873-9880, 1998). For these studies, the promoterdriving the expression of the antibodies is comprised of a CMV enhancer,the chicken beta-actin promoter and splice donor, and the rabbitbeta-globin splice acceptor (CAG). A schematic of the lentiviraltransfer vector is diagrammed in FIG. 2.

Lentivirus production: Vector production, concentration, p24 analysis,and titer assays were performed as previously described (Dull et al., J.Virol. 72:8463-8471, 1998). Briefly, vectors were prepared by transienttransfection in a 10 cm dish with 6.5 ug of pMDLg/pRRE, 2.5 ug ofpRSV-Rev, 3.5 ug of pMD2.VSVG-Env, and 10 ug of transfer vector. Vectorparticles were harvested after 24 hrs, pooled, passed through a 0.2 umcellulose acetate filter, and concentrated by ultracentrifugation for 2hrs 20 min at 19,500 rpm (50,000g) in a SW28 swinging bucket rotor.Pellets were resuspended in PBS containing 40 mg/ml lactose and storedin aliquots at −80° C. Detection of the gag p24 protein was evaluatedusing an Alliance HIV-1 p24 ELISA kit (Perkin Elmer).

AAV production: Recombinant AAV virus was prepared according to standardprocedures described in Snyder et al., 1996, In: Current Protocols inHuman Genetics, Seidman J S, (editor). John Wiley & Sons: New York;1-24. Briefly, sub-confluent 293 cells were co-transfected with thevector construct pAAV-CAG-KDR (2.13)-HF2AL, AAV helper plasmid pUC-ACGand Adeno helper plasmid pXX6 using the calcium phosphate method. Eighthours after transfection, media was replaced by fresh culture media andcells were incubated for 72 hr, at which point cells were harvested andlysed by three freeze/thaw cycles. Lysates were treated with Benzonase(EM Industries, Hawthorne, N.Y.) for 15 min at 37° C. to digest nucleicacids, and centrifuged to remove the cellular debris. The cleared celllysate was fractionated by ammonium sulfate precipitation and the rAAVvirions were isolated on two sequential CsCl gradients. The gradientfractions containing rAAV were dialyzed against sterile PBS containingCaCl₂ and MgCl₂, and stored at −80° C. AAV titers were calculated asgenomic equivalents, following DNase I and proteinase K treatment, bydot blot and by quantitative PCR as described in Harding et al., 2004Gene Ther (11): 204-213.

Transfection, selection and cloning: CHOD-cells were seeded at 3×10⁶ in10 cm plates 24 hr prior to transfection. The transfection of 12 ug ofDHFR-containing plasmid per plate was performed with Fugene 6 reagent(Roche Molecular Biochemical) according to the manufacturer's protocolin serum free OPTI-MEM I medium (Invitrogen). 5-6 hours posttransfection the medium was replaced with regular growth medium (50:50F12/DMEM medium supplemented with 2 mM L-glutamine, 10 ug /ml glycine,15 ug/ml hypoxanthine, 5 ug/ml thymidine and 10% FBS) and incubated at37° C., 5% CO₂. DHFR selection was carried out 48-72 hr posttransfection in IMDM medium (JRH) with 2 mM L-glutamine and 10% dialyzedFBS. 10 days post selection clones were picked into 96-well plates, andduplicate plates were made 2-3 days later. 24-hour supernatant wascollected from one of the duplicate plates and subjected to ELISA forantibody production and viable cell numbers in each well were determinedby CCK-8 proliferation assay (Dojindo). The data from ELISA and CCK-8assay was used to determine the pg/cell/day antibody production level ofeach clone. Clones>1 pg/cell/day were expanded from the second plate forfurther characterization. Clones or populations that were selected forMethotrexate (MTX) amplification were started at 5×10⁵ cells per 10 cmplate in MTX containing medium. MTX concentration was increased from 25nM to 50 nM, 100 nM, etc. Cells in each selection were passaged 2-3times before moving into a higher concentration of selective medium anda population of cells was banked (frozen) after each selection.

Lentivirus infection and cloning: CHOD-cells were seeded at 1×10⁵ cellsper well in 6-well plates with 2 ml culture medium containingappropriate amount of lentivector and polybrene at 8 ug/ml. Medium waschanged 24 hr post infection. Once the cells were confluent they wereexpanded to a 10 cm plate. Successive rounds of infections wereperformed at 2-7 day intervals. Populations were subcloned by limitingdilution. Clones were picked and screened as described in thetransfection method.

Methods for making clones: In order to determine the relativeeffectiveness of different vector systems, a comparison of variousmethods of making cell lines that express a rat anti-mouse VEGFR2antibody (DC 101) and a human IgG1 anti-KDR antibody (2.13) wereevaluated including:

(1) transfection using a plasmid which includes a 2A sequence whereinheavy and light chain are expressed under control of a single promoter,followed by amplification with methotrexate;

(2) transfection using a plasmid which includes dual promoters toexpress antibody heavy and light chains, followed by amplification withmethotrexate;

(3) infection with an AAV construct which includes a 2A sequence whereinheavy and light chains are expressed under control of a single promoter;and

(4) infection with a lentivirus construct which includes a 2A sequencewherein heavy and light chains are expressed under control of a singlepromoter.

Example 2 Comparing Different Methods for Making StableAntibody-Producing Cell Lines

Stable antibody expressing cell lines were made in CHOD-cells by Fugene6 transfection with a dual promoter anti-KDR plasmid or a 2A-anti-KDRplasmid as described above. Alternatively, CHOD-cells were infected witheither an AAV-2A-anti-KDR vector or a Lenti-2A-anti-KDR vector. In thisexample CHOD-cells were infected 5 times (5×) at one-week intervals withlenti-2A-KDR vector supernatants containing 500 ng p24. AAV-2A-KDRinfections were performed 3 times at an MOI of 10⁵ particles/cell. Thetransfected populations, lenti 4× and 5× infected populations, and AAVtransduced populations were all subcloned. Clones were picked, screenedand expanded as described above. Table 2 compares these four methods.

TABLE 2 Evaluation of anti-KDR antibody expressing clones produced bydifferent methods. Total clones >1 pg/cell/ >10 pg/cell/ >10 pg/cell/Method examined day 96-well day 6-well day 10-cm Transfection 373 2 0 02A-KDR Transfection 373 11 0 0 dual-KDR AAV-2A-KDR 800 2 0 0 Lenti-2A-KDR 260 260 >41 20 4X pop Lenti-2A-KDR 180 180 >14 6 5X pop

The results in Table 3 indicate that transfection of CHOD-cells with aplasmid which comprises an antibody heavy and light chain codingsequence operatively linked to a single promoter and further including aself processing 2A sequence did not show an advantage over dual promotercontrolled expression of antibody heavy and light chain coding sequencesfollowing transfection and screening. Alternatively, four or five roundsof transfection/transduction of CHOD-cells with lentivector constructswhich comprise an antibody heavy and light chain coding sequenceoperatively linked to a single promoter and further including a selfprocessing 2A sequence resulted in transduction of 100% of clones,yielding higher numbers of clones with significantly greater levels ofantibody production compared to the other methods. This approach alsoreduced the number of clones necessary to screen and significantlyreduced the time necessary for isolation of high expressing clones (FIG.3). Multiple rounds of transduction with a lentivector also increasedthe copy number in a producer cell resulting in high level proteinexpression. The twelve lenti clones with the highest expression levelswere plated at 1×10⁷ cells/10 cm plate and evaluated for antibodyexpression at 31° C. (Table 3). These data show that all of theindividual clones evaluated express high levels of antibody.

TABLE 3 Lenti-2A-KDR CHOD- expressing clones. # of clone # infectionspg/cell/day 22 4X 18.2 30 4X 26.2 61 4X 18.6 62   4X 1 5.0 70 4X 18.2 874X 26.7 15 5X 20.1 65 5X 21.9 66 5X 16.0 80 5X 10.7 88 5X 10.9 89 5X 9.9

A similar experiment comparing methods of producing clones was performedusing the rat anti mouse DC101 antibody. CHOD-transfections wereperformed as described above to compare protein expression followingtransfection with the 2A DC101 plasmid relative to the dual promoterDC101 plasmid. Alternatively, five serial infections of CHOD-cells wereperformed at 1-week intervals with the lenti-2A-DC101 vector (200 ngp24/infection). In all three cases the clones were isolated and analyzedfor antibody expression. Table 4 compares the expression levels of the2A-DC101 plasmids with the dual promoter (H+L) plasmids. There was noapparent advantage to the 2A construct in transfection experiments. Ingeneral, expression levels of the anti-DC101 antibody are higher thanthe anti-KDR antibody. The antibody expression in the lenti-2A-DC101population was much higher than the transfected populations. Again,fewer clones had to be screened due to higher antibody expressionlevels, and the greater frequency of positive clones as compared to theother systems tested. For example, when expanded to 6 well plates all ofthe lentiviral clones produced greater than 1 p/c/d of antibody. Ten ofthe highest producing lenti-2A clones from 6-well plates were expandedto 10-cm plates for further analysis (Table 6). While only 8/10 clonesexceeded 10 p/c/d, two of the clones, #45 and 51, were exceptionallyhigh producers, again demonstrating that serial rounds of transductionwith lenti 2A vectors results in the need to perform screening of fewerclones in order to obtain high expressing clones. This method alsoeliminates the need for an amplification step, e.g., with DHFR.

TABLE 4 Comparison of antibody expression levels in CHOD- clonestransfected with 2A-DC101 or dual promoter DC101 plasmids. # clones #clones Population >1 p/c/d >1 p/c/d Plasmid pg/cell/day Total clones96-well 6-well H + L DC101 1.7 400 137 120/137 2A-DC101 0.55 400 8475/84 Lenti 5X 8.17 130 130 60/60

TABLE 5 Antibody expression in CHOD- clones infected 5X withLenti-2A-DC101. Clone # p/c/d 8 13.69 22 13.77 31 15.78 38 8.49 39 7.5745 45 47 9.96 51 40 55 14.09 59 11.48

The number of integrated genomic copies in cells transfected with thelentiviral vector comprising the immunoglobulin 2A construct encodingthe DC101 antibody was also examined by Southern Blot analysis (FIG. 5).Genomic DNA was isolated from naive CHO cells and from two 5× transducedclones that express approximately 20-40 pg/cell/day (Clones 45 and 51).The genomic DNA was digested using the restriction enzyme EcoRI usingstandard conditions and resolved by electrophoresis on a 1% agarose gelin the presence of known, increasing genomic amounts of control DNA toestimate copy number. The resolved DNA fragments were transferred tonylon filter for further analysis. The filter hybridized to a 2.2 Kbradiolabeled DNA fragment comprising the full-length nucleotide sequenceencoding the DC101 antibody. The position of the bound probe wasvisualized using autoradiography and quantitated using a phosphorimager(Molecurar Dynamics, Sunnyvale, Calif.).

As shown in FIG. 5, the radiolabeled probe hybridizes to a single 2.2 Kbfragment containing the nucleotide sequence encoding the DC101 antibody.Clones 45 and 51 also exhibit only a single 2.2 Kb band and compriseapproximately 33 genomic copies of the lentiviral vector construct. Thisnumber is consistent with the Taq-man results presented above. Thus,within a 5-week period, a high producer cell line comprising 33 copiesof the antibody coding sequences may be prepared without drug-basedamplification.

CHOD-cells were transfected with 200 ng of p24 of the lentiviral vectorencoding DC101 4×, 7× or 9× at 3 day intervals. Populations from thetransfected cells were subcloned and approximately 100-160 clones wereexpanded into 96 well plates and the supernatants screened by ELISA todetermine DC101 expression levels. Cell number was determined using aCCK8 assay. Approximately 50 high expressing clones were chosen,expanded in 6-well plates and the amount of antibody produced(pg/cell/day) was determined for each clone (FIG. 6). As the number oftransfections was increased, there was a concomitant increase in thenumber of cells that express increasing levels of antibody, e.g.,compare pg/cell/day of antibody production for 4× and 9× clones (FIG.6). Furthermore, approximately 50% of the cells transfected in the 9×population expressed greater that 30 pg/cell/day thereby greatlyreducing the time and number of clones required to be screened toidentify cell lines capable of expressing commercially-relevant amountsof recombinant antibody.

Example 3 Demonstration of Antibody Expression Levels from DifferentCell Types Following Transduction with Lentiviral Constructs

Antibody expression levels were evaluated for lenti human anti-KDR andlenti rat anti-VEGFR2 (DC101) while looking at a number of variables:different cell types, different promoters, and different lenticonstructs. The different cell types examined were CHOD-cells, PerC6cells and HuH7 cells. The different promoters were CAG, CMV and MND.

For the assays CHOD-cells, PerC6 cells, or HuH7 cells were seeded at1×10⁵ cells per well in 6-well plates containing 2 ml of culture mediumincluding an appropriate amount of lentivector and polybrene at 8 ug/ml.Infection was allowed to proceed as described above. The medium waschanged 24 hr post infection. Cells were maintained in 6-well platesuntil they reached 80% confluence. The medium was then refreshed and at24 hr and supernatant collected from each well. The cell numbers in eachwell were determined by hemocytometer. The antibody expression level wasdetermined by ELISA.

FIG. 4 compares the antibody production from three cell lines (CHOD-,PerC6, HuH7) infected with lenti-2A-anti-DC101. FIG. 7 demonstrates thatantibody is expressed in the same cell lines following infection withlenti-2A-anti-KDR using a CMV promoter.

Example 4 Alteration in the Amount of Lentivector and Changes in theInfection Intervals can Increase Antibody Expression Levels and DecreaseTime for Identifying Clones

CHOD-cells were serially infected with lenti-2A-DC101 vector.Transductions done at one week intervals were compared with transductionis done at 2-3 day intervals. Infections were performed as describedabove. After each infection, the populations were saved, expanded, andassayed for antibody expression as described. Additionally, each cellpopulation was analyzed for the number of lenti copies/cell by TaqManPCR (Table 7).

TABLE 6 Comparison of antibody expression and lentivector copy number inCHOD- populations infected with Lenti-2A-DC101 at different timeintervals. Experiment #1 Experiment #2 Day of Lenti Population Day ofLenti Population infection copies/cell pg/cell/day infection copies/cellpg/cell/day P2ng/ml  50 1 7 1.60 * * * 100 1 37 2.26 * * * 200 (1X) 1 203.62 1 20 4.58 200 (2X) 8 40 7.07 3 41 7.62 200 (3X) 15 36 7.50 5 588.05 P4ng/ml 200 (4X) 22 43 8.64 8 97 9.46 200 (5X) 29 77 8.17 11 9411.30 200 (6X) * * * 14 75 12.54 200 (7X) * * * 17 145 14.49 200(8X) * * * 20 135 13.98 200 (9X) * * * 23 125 15.09

Taken together, the results suggest that successive transductions withlentivectors increases the number of antibody producer clones thatgenerate high levels of antibodies. Integrated lentivector copiesincrease with each transduction and correlates with increasing antibodyexpression in the populations. It is interesting to note that decreasingthe time between infections had a positive effect on antibody expressionlevels. Additionally, by decreasing the interval between infections from1 week to 2-3 days, antibody expression was increased approximately2-fold and the overall infection process was decreased by a week.

Example 8 Removal of Extra Amino Acids Derived from Furin Cleavage Siteat C-Terminus by Carboxypeptidases

It has been shown that after furin cleavage newly exposed basic aminoacids at the C-terminus of proteins can be removed by carboxypeptidases,we hypothesized that all additional amino acid derived from the furincleavage site at the C-terminus of antibody heavy chain can be removedby using a furin cleavage site that consists of exclusively basic aminoacids, i.e. R or K. In testing a number of furin cleavage sites with allbasic amino acids, one construct was developed wherein the last aminoacid K at the C-terminus of antibody heavy chain was deleted and theantibody heavy and light chains were linked with a furin cleavage siteRKRR (SEQ ID NO: 15) and a 2A sequence. The plasmid DNA was transfectedinto CHO cells in 10 cm tissue culture dish and the antibody protein waspurified from the supernatant by protein A affinity chromatography. Thepurified IgG protein was separated in SDS-PAGE and the heavy chain bandwas sliced out from the gel. The heavy chain protein was then cleaved byCyanogen Bromide (CNBr) and the resulting peptides were analyzed by massspectrometer. Mass spectral analysis showed a strong peak thatcorresponds to the C-terminal fragment of the antibody heavy chain (FIG.8). No peaks were observed in the spectrum that would represent theC-terminal fragment plus any amino acid(s) derived from the furincleavage site. This data strongly suggested that the RKRR (SEQ ID NO:15) site facilitates efficient removal of extra amino acids at theC-terminus of the antibody heavy chain in HF2AL constructs, resulting inexpression of the antibody heavy chain without any extra amino acidresidues.

Example 9 Stable Long-Term Expression of Lentivectors in HumanPancreatic and Mouse CT26 Cells

Lentiviral vectors expressing human GMCSF from a CAG promoter wereprepared by transient transfection, as described. Pancreatic cells werecultured and 3×10⁴ cells were spinoculated with 1 ml of lentivirus and 8ug/ml polybrene for 4 hrs at 3400 rpm, then plated in a 6 well platewith fresh medium. A total of three infections were performed at 2 weekintervals. The populations from each infection were labeled “1×, 2×, and3×.” The 3× population was dilution cloned, and 1280 clones were pickedand transferred to 96 well plates. Supernatants from these clones werescreened for GM-CSF expression by ELISA. Nearly all clones were positivefor human GM-CSF expression, and 88 clones were expanded to 6-wellplates and re-assayed. Thirteen clones expressing 500-2500 ng/10⁶cells/24 hr GM-CSF were expanded to 10 cm plates, reassayed again, andput into a stability study. For the stability study the cells weremaintained in continuous culture for 12 weeks with GM-CSF expressionlevels checked at 3 week intervals. FIG. 9 demonstrates that in 13clones lentivector GM-CSF expression was stable for 12 weeks ofcontinuous culture in medium that did not contain any selection.

In a second lentivector expression stability study, long-term expressionof mouse GM-CSF-producing clones was evaluated using CT26 cells (mouseadenocarcinoma cells) transduced with lentiviral vector expressing mouseGM-CSF. The CT26 cells were spinoculated and cloned as described. Fiveclones expressing GM-CSF were selected for a stability study. At weeks1, 3, 5, 7 and 9, the cells were assayed for GM-CSF expression by ELISA.The results shown in FIG. 10 show that 5 clones exhibited stable GM-CSFexpression over the course of the study.

Both the pancreatic and CT26 GM-CSF expression experiments demonstratethat lentivector generate clones are stable in long-term culture.

Example 10 Stable Long-Term Expression Of Lentivectors In CHO-S Cells

Lentiviral vectors expressing human DC101-IgG1 or KDR-IgG4 from a CAGpromoter were prepared. The lentivector construct contained a 33 aminoacid sequence from Foot and Mouth Disease Virus (FMDV) designated as2A14 (SEQ ID NO: 9) and an optimized furin sequence RKRR (SEQ ID NO:15), referred to herein as the “Lenti-DC101 vector”. Suspension adapted,serum-free CHO-S cells or suspension adapted NSO cells were transfectedwith the lentivector construct. The DC101 antibody coding sequence wasmodified so that the C terminal contains a methionine that can becleaved for Mass Spec analysis.

CHO-S cells were serially infected with 200 ng P24 of the Lenti-DC101vector at 2 to 3 day intervals. The populations were assayed for DC101expression by ELISA (Table 1), and the data demonstrate that a steadyincrease in antibody production resulted following each infection, withno impact on the doubling times.

TABLE 7 DC101 Expression from CHO-S Cells Infected withLenti-DC101-furin/2A14 by ELISA. # of infections Doubling time (hrs)pg/cell/day 1x 18 7 2x 18 9 3x 19 19 4x 19 18 5x 17 16 6x 18 22 7x 19 24

Populations of cell that were infected 4× and 7×, respectively, weresubcloned and analyzed for DC101 production. When compared, the clonesderived from the population infected 7× generally had higher levels ofDC101 expression than the clones from the population infected 4×. (Table2).

TABLE 8 DC101 expression by CHO-S cells infected 4x or 7x withLenti-DC101-furin/2A14. Range of pg/cell/day 4X population 7X population1 1 0 10 18 4 20 19 13 30 5 12 40 3 7 50 0 3 60 0 3 70 0 3 80 0 1 90 0 0100 0 0 110 0 0 120 0 1 Total Clones 46 47

A panel of clones isolated from the 2', 3× and 4× infected populationswere subjected to continuous culture in 10 ml shaker flasks for 12 weeksand evaluated at one week intervals for stability of DC101 production.The clones were found to be stable (Table 3).

TABLE 9 Long Term DC101 expression by CHO-S cells infected withLenti-DC101-furin/2A14. Clones: pg/cell/day weeks 4x-4 4x-3 4x-2 4x-13x-3 2x-8 2x-5 1 61.8 71.7 45.6 34.9 22.4 28.7 23.3 2 76.2 59.3 51.034.8 24.1 25.6 23.2 3 89.3 79.0 101.2 66.5 41.9 37.8 45.4 4 67.2 73.658.0 35.9 43.7 65.4 62.0 5 61.8 75.7 67.9 21.1 23.8 24.8 16.2 6 99.562.6 49.8 47.4 30.4 30.8 23.3 7 58.3 57.4 56.9 45.5 24.4 24.4 23.5 863.4 44.2 54.7 23.9 35.3 21.2 22.2 9 62.0 36.9 57.4 23.2 20.7 27.6 22.710 64.2 42.7 57.3 30.0 20.8 18.6 23.5 11 54.6 48.1 38.3 24.6 22.9 22.928.0 12 55.7 52.4 31.4 26.2 24.5 27.8 33.9

the doubling times stabilized at less than 20 hours (Table 4).

TABLE 10 Doubling Time of DC101 expressing CHO-S cells infected withLenti-DC101-furin/2A14. Clones: Doubling time (hrs) weeks 4x-4 4x-3 4x-24x-1 3x-3 2x-8 2x-5 1 17.1 25.5 16.2 15.5 16.6 16.2 14.0 2 21.2 30.117.7 16.7 17.1 16.7 16.2 3 21.2 35.7 21.2 21.2 18.1 18.1 18.7 4 21.229.1 21.2 16.2 22.4 29.4 22.9 5 17.1 19.4 16.2 15.5 16.4 15.9 15.2 618.1 19.4 15.8 16.2 15.9 15.9 14.9 7 20.2 19.4 16.6 18.1 16.2 15.5 14.98 17.2 18.1 16.6 15.9 17.8 15.2 15.9 9 18.1 17.2 17.1 16.2 16.4 15.215.5 10 16.6 17.1 17.2 14.9 16.0 15.1 15.3 11 19.7 16.6 16.2 15.7 15.615.5 15.9 12 19.1 18.1 15.9 16.2 16.2 15.9 14.7

Clones 4×-3 and 4×-4 were analyzed for genetic stability by Southernblot. DNA was extracted from both clones at weeks 1 and 7, digested withrestriction enzymes EcoR I or Nsi I overnight, run on a 1% agarose/TBEgel and probed with a 592 by fragment from the HC region. The standardis a serial dilution of EcoR I-digested DC101 plasmid in a background of10 mg of genomic CHO-S DNA. The EcoR I releases a 2.2 kb fragment inboth clones, and Clone 4×-4 appears to have several partial integrants.With both restriction enzymes the two clones have stable integrationpatterns from 1 to 7 weeks, demonstrating genetic stability.

Binding and neutralization assays were done to establish that antibodiesfrom these clones were biologically equivalent to antibody produced froma hybridoma. In the binding assays 96-well plates were coated withrecombinant Flk-1, incubated with serial dilutions of supernatants fromclone 7×-24, the DC101 hybridoma, or a control, and quantified withanti-rat IgG1 HRP. The results show that the antibodies bindequivalently (Table 5).

TABLE 11 Binding of DC101 expressed from CHO-S cells infected withLenti- DC101-furin/2A14 relative to DC101 Produced by a hybridoma. ng/mlMock CHO-S Hybridoma 1000 0.152 1.144 1.121 333 0.149 0.993 0.894 1110.152 0.616 0.724 37 0.121 0.486 0.585 12 0.120 0.412 0.403 4 0.1040.274 0.312 0 0.118 0.129 0.117

A neutralization assay was carried out using supernatants from Clone7×-24, a DC101 hybridona and a negative control. Each supernatant wasserially diluted and mixed with Flk-1, then incubated on 96-well platepre-coated with human VEGF and read with anti-Flk-1-HRP. The antibodiesgenerated from the 7×-24 clone neutralized the Flk-1 binding at a levelequivalent to the hybridoma-produced DC 101 antibody (Table 6).

TABLE 12 Neutralization Assay of DC101 antibody produced by clone 7X-24versus hybridoma-produced DC101. ng/ml Mock CHO-S Hybridoma 1000 1.2780.184 0.193 333 1.28 0.237 0.357 111 1.203 0.43 0.635 37 1.195 0.8260.834 12 1.134 0.945 0.931 4 1.152 1.043 0.992 0 1.089 1.069 1.105

The clones were tested for replication competent lentivirus by a TaqManassay for VSV.G sequences. To establish the sensitivity of the assay, acell line known to contain 1 copy of VSV-G envelope was spiked into abackground of CHO cells ranging from 10-10,000 cells. DNA samples wereprepared and assayed for the presence of VSV.G sequences. Thesensitivity of this assay allows for detection of one VSV.G sequence ina background of 10,000 cells. The three clones and the CHO-S controlcells were all negative for replication-competent lentiviral sequences.

TABLE 13 Sequences for Cell 164.1. (SEQ ID) SEQUENCE NO: 1LLNFDLLKLAGDVESNPGPSEQUENCE NO: 2 TLNFDLLKLAGDVESNPGP NO: 3LLKLAGDVESNPGP NO: 4 NFDLLKLAGDVESNPGP NO: 5 QLLNFDLLKLAGDVESNPGP NO: 6APVKQTLNFDLLKLAGDVESNPGP NO: 7 VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGPSEQUENCE NO: 8LLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP NO: 9EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP NO: 10Furin consensus sequence or site RXK(R)R NO: 11Factor Xa cleavage sequence or site:  IE(D)GR NO: 12Signal peptidase I cleavage sequence  or site: e.g., LAGFATVAQA NO: 13Thrombin cleavage sequence or site:  LVPRGS NO: 14 Furin site-RKKRNO: 15 Furin site-RKRR NO: 16 Furin site-RRKR NO: 17 Furin site-RRRR

1. A lentivector for expression of a recombinant immunoglobulin,comprising: in the 5′ to 3′ direction, a promoter operably linked to thecoding sequence for a first chain of an immunoglobulin molecule or afragment thereof, an additional proteolytic cleavage site, a sequenceencoding a self-processing cleavage site and the coding sequence for asecond chain of an immunoglobulin molecule or fragment thereof, whereinthe sequence encoding the self-processing cleavage site is insertedbetween the coding sequence for the first chain and the coding sequencefor the second chain of said immunoglobulin molecule.
 2. The lentivectoraccording to claim 1, wherein the sequence encoding the self-processingcleavage site comprises a 2A sequence.
 3. The lentivector according toclaim 2, wherein the 2A sequence is a Foot and Mouth Disease Virus(FMDV) sequence.
 4. The lentivector according to claim 3, wherein the 2Asequence encodes an oligopeptide comprising amino acid residuesLLNFDLLKLAGDVESNPGP (SEQ ID NO: 1) or TLNFDLLKLAGDVESNPGP (SEQ ID NO:2)or EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:9).
 5. The lentivectoraccording to claim 3, wherein the coding sequence for the first chain ofsaid immunoglobulin molecule or a fragment thereof encodes animmunoglobulin heavy chain.
 6. The lentivector according to claim 3,wherein the coding sequence for the first chain of said immunoglobulinmolecule or a fragment thereof encodes an immunoglobulin light chain. 7.The lentivector according to claim 5, wherein the coding sequence is thefull length coding sequence of an immunoglobulin heavy chain.
 8. Thelentivector according to claim 6, wherein the coding sequence is thefull length coding sequence of an immunoglobulin light chain.
 9. Thelentivector according to claim 1, wherein said additional proteolyticcleavage site is a furin cleavage site with the consensus sequence RKRR(SEQ ID NO: 15).
 10. The lentivector according to claim 3, wherein thepromoter is selected from the group consisting of an elongation factor1-alpha promoter (EF1 a) promoter, a phosphoglycerate kinase-1 promoter(PGK) promoter, a cytomegalovirus immediate early gene promoter (CMV), achimeric liver-specific promoter (LSP) a cytomegalovirusenhancer/chicken beta-actin promoter (CAG), a tetracycline responsivepromoter (TRE), a transthyretin promoter (TTR), a MND promoter, a simianvirus 40 promoter (SV40) and a CK6 promoter.
 11. The lentivectoraccording to claim 10, wherein said promoter is a CAG hybridpromoter/enhancer.
 12. The lentivector according to claim 10, whereinsaid promoter is a CMV promoter/enhancer.
 13. The lentivector accordingto claim 3, further comprising a signal sequence.
 14. The lentivectoraccording to claim 3, wherein said heavy and light chain immunoglobulincoding sequences are expressed in a substantially equimolar ratio. 15.The lentivector according to claim 11, wherein said lentivectorcomprises a CAG promoter operably linked to the coding sequence for afirst chain of an immunoglobulin molecule, a sequence encoding aself-processing cleavage site and the coding sequence for a second chainof an immunoglobulin molecule, wherein the sequence encoding saidself-processing cleavage site is inserted between the coding sequencefor the first chain and the coding sequence for the second chain of saidimmunoglobulin molecule.
 16. A producer cell transduced with the vectorof claim
 11. 17. A producer cell transduced with the vector of claim 12.18. A producer cell transduced with the vector of claim
 14. 19. Arecombinant immunoglobulin molecule produced by a producer cell infectedwith the lentivector of claim
 11. 20. A recombinant immunoglobulinmolecule produced by a producer cell infected with the lentivector ofclaim
 12. 21. A recombinant immunoglobulin molecule produced by aproducer cell infected with the lentivector of claim
 14. 22-39.(canceled)